Metalloprotease proteins

ABSTRACT

This invention relates to proteins termed INSP005 a  and INSP005 b , herein identified as secreted proteins, in particular members of the metalloprotease family and to the use of these proteins and nucleic acid sequences from the encoding genes in the diagnosis, prevention, and treatment of disease.

CROSS-REFERENCE TO RELATED APPLICATION

This application is the National Stage of International ApplicationNumber PCT/GB2003/005664, filed Dec. 23, 2003, which is herebyincorporated by reference herein in its entirety, including any figures,tables, nucleic acid sequences, amino acid sequences, or drawings.

This invention relates to novel proteins, termed INSP005a and INSP005b,herein identified as secreted proteins, in particular members of themetalloprotease family and to the use of these proteins and nucleic acidsequences from the encoding genes in the diagnosis, prevention andtreatment of disease.

All publications, patents and patent applications cited herein areincorporated in full by reference.

BACKGROUND

The process of drug discovery is presently undergoing a fundamentalrevolution as the era of functional genomics comes of age. The term“functional genomics” applies to an approach utilising bioinformaticstools to ascribe function to protein sequences of interest. Such toolsare becoming increasingly necessary as the speed of generation ofsequence data is rapidly outpacing the ability of research laboratoriesto assign functions to these protein sequences.

As bioinformatics tools increase in potency and in accuracy, these toolsare rapidly replacing the conventional techniques of biochemicalcharacterisation. Indeed, the advanced bioinformatics tools used inidentifying the present invention are now capable of outputting resultsin which a high degree of confidence can be placed.

Various institutions and commercial organisations are examining sequencedata as they become available and significant discoveries are being madeon an on-going basis. However, there remains a continuing need toidentify and characterise further genes and the polypeptides that theyencode, as targets for research and for drug discovery.

SECRETED PROTEIN BACKGROUND

The ability for cells to make and secrete extracellular proteins iscentral to many biological processes. Enzymes, growth factors,extracellular matrix proteins and signalling molecules are all secretedby cells. This is through fusion of a secretory vesicle with the plasmamembrane. In most cases, but not all, proteins are directed to theendoplasmic reticulum and into secretory vesicles by a signal peptide.Signal peptides are cis-acting sequences that affect the transport ofpolypeptide chains from the cytoplasm to a membrane bound compartmentsuch as a secretory vesicle. Polypeptides that are targeted to thesecretory vesicles are either secreted into the extracellular matrix orare retained in the plasma membrane. The polypeptides that are retainedin the plasma membrane will have one or more transmembrane domains.Examples of secreted proteins that play a central role in thefunctioning of a cell are cytokines, hormones, extracellular matrixproteins (adhesion molecules), proteases, and growth and differentiationfactors. Description of some of the properties of these proteinsfollows.

Proteases are enzymes that irreversibly hydrolyse amide bonds inpeptides and proteins. Proteases are widely distributed and are involvedin many different biological processes, from activation of proteins andpeptides to degradation of proteins. Despite the fact that proteaseshave been shown to be involved in many different diseases, drugstargeted to proteases are still rare in pharmacy, although inhibitors ofangiotensin converting enzyme (ACE) have been among the most successfulantihypertensive drugs for several years. Proteases have recentlyreceived substantial publicity as valuable therapeutic targets followingthe approval of HIV protease inhibitors.

Proteases can be divided in large Families. The term “Family” is used todescribe a group of proteases in which each member shows an evolutionaryrelationship to at least one other member, either throughout the wholesequence or at least in the part of the sequence responsible forcatalytic activity. The name of each Family reflects the catalyticactivity type of the proteases in the Family. Thus, serine proteasesbelong to the S family, threonine proteases belong to the T family,aspartyl proteases belong to the A family, cysteine proteases belong tothe C family and metalloproteinases belong to the M family.Metalloproteases and Serine proteases are commonly found in theextracellular matrix.

Metalloproteases (M Family):

Metalloproteases can be divided in 2 major groups depending on thepresence or absence of a the Zinc binding motif (HEXXH).

1.1 Presence of HEXXH motif(22 families): Prosite number: PDOC00129

Families with Interesting Members:

-   M2: Peptidyl-dipeptidase A (Angiotensin I Converting Enzyme: ACE)-   M13: Neprilysin (Enkephalinase A=neutral endopeptidase=NEP),    Endothelial Converting Enzyme (ECE)-   M10B: Matrixin (Matrix Metalloproteases=MMPs)-   M12B: Reprolysin (ADAM-10; ADAM-17=TNF-alpha Converting    Enzyme=TACE)/Desintegrin (other ADAM proteases). The ADAMs are a    large, widely expressed and developmentally regulated family of    proteins with multiple potential functions in cell-cell and    cell-matrix interactions. Among them TACE represents a new emerging    target for arthritis disease.-   M41: This family contains ATP-dependent metalloproteases: FtsH,    proteasome proteins.

One of the largest therapeutically interesting group ofmetalloproteinases is the Matrix Metalloproteinases family (MMPs).Matrix metalloproteinases are a family of Zinc containing enzymes thatare responsible for the remodeling of extracellular matrix throughoutthe body. They have been shown to be involved in cancer (increaseinvasiveness, effects on new blood vessel), and in arthritis(involvement in cartilage degradation (Dahlberg, L., et al., ArthritisRheum. 2000 43(3):673-82) and also TNF-alpha conversion (Hanemaaijer,R., et al., J Biol Chem. 1997 272(50):31504-9, Shlopov, B. V., et al.,Arthritis Rheum. 1997 40(11):2065-74)). Indeed, different MMPs have beenshown to be overexpressed in diseases such as arthritis (Seitz, M., etal., Rheumatology (Oxford). 2000 39(6):637-645, Yoshihara, Y., et al.,Ann Rheum Dis. 2000 59(6):455-61, Yamanaka, H., et al., Lab Invest. 200080(5):677-87, Jovanovic, D. V., et al., Arthritis Rheum. 2000 May;43(5):1134-44, Ribbens, C., et al., J Rheumatol. 2000 27(4):888-93) andcancer (Sakamoto, Y., et al., Int J Oncol. 2000 17(2):237-43, Kerkela,E., et al., J Invest Dermatol. 2000 114(6):1113-9, Fang, J., et al.,Proc Natl Acad Sci USA. 2000 97(8):3884-9, Sun, Y., et al., J Biol Chem.2000 275(15):11327-32, McCawley, L. J., et al., Mol Med Today. 20006(4):149-56, Ara, T., et al., J Pediatr Surg. 2000 35(3):432-7,Shigemasa, K., et al., Med Oncol. 2000 17(1):52-8, Nakanishi, K., etal., Hum Pathol. 2000 31(2):193-200, Dalberg, K., et al., World J Surg.2000 24(3):334-40). Inhibitors of these enzymes have been suggested aspotential therapeutic agents for the use in the treatment of both cancerand arthritis. More recently it has been shown that MMPs may also have arole in the release of soluble cytokine receptors, growth factors andother cell mediators, suggesting that selective MMPs inhibitors may havewider therapeutic applications than previously proposed.

MMPs have been divided in 4 families based on amino-acid sequencehomologies of their domain structure, other than the catalytic region.

Minimal domain family: matrilysin (PUMP-1, MMP-7) cleaves proteoglycan,laminin and fibronectin

Hemopexin domain family:

Collagenases: unique ability to cleave fibrillar collagen. The role ofcollagenases in cartilage degradation, make them attractive targets forthe treatment of rheumatoid and osteo-arthritis.

-   -   collagenases fibroblast collagenase (interstitial collagenase,        MMP-1)    -   neutrophil collagenase (MMP-8)    -   collagenase-3 (MMP-13)

Metalloelastase: MME (MMP-12)

-   Stromelysin-1 (MMP-3), 2 (MMP-10) and 3 (MMP-11). MMP-11 is excreted    as an active form and it's function could be to activate other MMPs.    Fibronectin domain family: degrades a large number of matrix    substrates (gelatin, elastin, type IV collagen)-   Gelatinase A (MMP-2); beside it's involvement in cancer (tumor    invasiveness), it is proposed as a potential target for the    discovery of antiplatelet agent as it may play an important role in    platelet activation.-   Gelatinase B (MMP-9)    Transmembrane domain family:-   MT-1-MMP, MT-4-MMP, MMP-14, MMP-17

A lot of studies concerning the different specificities of MMPs andtheir relative involvement in some diseases are on going.

1.2 Absence of HEXXH motifs (18 families):

Families with interesting members:

-   M24A: Methionyl aminopeptidase, type 1 (including procaryotic and    eucaryotic MAP-1)/Prosite number: PDOC00575-   M24C: Methionyl aminopeptidase, type 2 (including eucaryotic    MAP-2)/Prosite number: PDOC00575

TABLE 1 Summary of metalloproteases and their function Protease EC namenumber Biological function Disease associated Regulation MMP-123.4.24.65 MMPs function; elastin degradation; involvement in lungenhanced expression in process TNF-alpha; convert plasminogen disorders,emphyscma, cystic some skin diseases to angiotensin fibrosis MMP-23.4.24.24 MMPs function cancer overexpression in colorectal cancerADAM-12 3.4.24 cell-cell, cell-matrix interaction up-regulated inseveral human carcinomas TACE 3.4.24.? Processing of the membrane boundTNF- inflammation, rheumatoid up-regulated in arthritis alpha and othercell bound molecule arthritis, neuroimmunological affected cartilagediseases ACE 3.4.15.1 production of angiotensin II hypertension ECE-13.4.24.71 process the precursor of the cardiovascular vasoconstrictorendothelin NEP 3.4.24.11 cleaves neuropeptides, hormones andcardiovascular, arthritis (?) immune mediator FtsH ? protein secretion,assembly, degradation, bacterial infections — cell cycle, stressresponse Deformylase 3.5.1.31 removes the formyl group from N- bacterialinfections — terminal from newly synthesized proteins Proteasome3.4.99.46 protein degradation, antigen presentation cancer

Metalloproteases are implicated across a wide variety of therapeuticareas. These include respiratory diseases (Segura-Valdez, L., et al.,Chest. 2000 117(3):684-94, Tanaka, H., et al., J Allergy Clin Immunol.2000 105(5):900-5, Hoshino, M., et al., J Allergy Clin Immunol. 1999104(2 Pt 1):356-63, Mautino, G., et al., Am J Respir Crit Care Med. 1999160(1):324-30, Dalal, S., et al., Chest. 2000 117(5 Suppl 1):227S-8S,Ohnishi, K., et al., Lab Invest. 1998 78(9):1077-87), cardiovasculardisease (Taniyama, Y., et al., Circulation. 2000 102(2):246-52, Hong, B.K., et al., Yonsei Med J. 2000 41(1):82-8, Galis, Z. S., et al., ProcNatl Acad Sci USA. 1995 92(2):402-6), bacterial infections (Scozzafava,A., et al., J Med Chem. 2000 43(9):1858-65, Vencill, C. F., et al.,Biochemistry. 1985 24(13):3149-57, Steinbrink, D. R, et al., J BiolChem. 1985 260(5):2771-6, Lopez-Boado, Y. S., et al., J Cell Biol. 2000148(6):1305-15, Chang, J. C., et al., Thorax. 1996 51(3):306-1 1,Dammann, T., et al., Mol. Microbiol. 6:2267-2278 (1992), Wassif, C., etal., J. Bacteriol. 177 (20), 5790-5798 (1995), oncology (Sakamoto, Y.,et al., Int J Oncol. 2000 17(2):237-43, Kerkela, E., et al., J InvestDermatol. 2000 114(6):1113-9, Fang, J., et al., Proc Natl Acad Sci USA.2000 97(8):3884-9, Sun, Y., et al., J Biol Chem. 2000 275(15):11327-32,McCawley, L. J., et al., Mol Med Today. 2000 6(4):149-56, Ara, T., etal., J Pediatr Surg. 2000 35(3):432-7, Shigemasa, K., et al., Med Oncol.2000 17(1):52-8, Nakanishi, K., et al., Hum Pathol. 2000 31(2):193-200,Dalberg, K., et al., World J Surg. 2000 24(3):334-40), and inflammation(rheumatoid and osteo-arthritis (Ribbens, C., et al., J Rheumatol. 200027(4):888-93, Kageyama, Y., et al., Clin Rheumatol. 2000 19(1):14-20,Shlopov, B. V., et al., Arthritis Rheum. 2000 January; 43(1):195-205)).

Metalloproteases are also implicated in the physiology and pathology ofsexual reproduction, and have been implicated in therapies associatedwith modulating chorion status, the zona reaction, the formation offertilisation membranes, contraception and infertility (Shibata et al.(2000) J. Biol. Chem vol. 275, No. 12 p 8349)

Accordingly, identification of novel metalloproteases is of extremeimportance in increasing understanding of the underlying pathways thatlead to certain disease states in which these proteins are implicated,and in developing more effective gene or drug therapies to treat thesedisorders.

THE INVENTION

The invention is based on the discovery that the INSP005a and INSP005bproteins function as secreted protease molecules and moreover assecreted protease molecules of the metalloprotease family. Preferably,the INSP005a and INSP005b proteins are members of thechoriolysin/astacin-like family of metalloproteases.

In one embodiment of the first aspect of the invention, there isprovided a polypeptide which:

-   (i) comprises the amino acid sequence as recited in SEQ ID NO:14;-   (ii) is a fragment thereof having function as a secreted protein of    the metalloprotease class or having an antigenic determinant in    common with the polypeptides of (i); or-   (iii) is a functional equivalent of (i) or (ii).

Preferably, a polypeptide according to this embodiment consists of thesequence recited in SEQ ID NO:14. The polypeptide having the sequencerecited in SEQ ID NO:14 is referred to hereafter as “the INSP005apolypeptide”.

In a second embodiment of the first aspect of the invention, there isprovided a polypeptide which:

-   (i) comprises the amino acid sequence as recited in SEQ ID NO:34 or    SEQ ID NO:36;-   (ii) is a fragment thereof having function as a secreted protein of    the metalloprotease class or having an antigenic determinant in    common with the polypeptides of (i); or-   (iii) is a functional equivalent of (i) or (ii).

Preferably, a polypeptide according to this embodiment consists of thesequence recited in SEQ ID NO:34 or SEQ ID NO:36. The polypeptide havingthe sequence recited in SEQ ID NO:34 is referred to hereafter as “theINSP005b polypeptide”.

Although the Applicant does not wish to be bound by this theory, it ispostulated that the first 23 amino acids of the INSP005b polypeptideform a signal peptide. The nucleotide sequence encoding the postulatedINSP005b mature polypeptide, and the amino acid sequence of the INSP005bmature polypeptide, are recited in SEQ ID NO:35 and SEQ ID NO:36,respectively. The polypeptide having the sequence recited in SEQ IDNO:36 is referred to hereafter as “the INSP005b mature polypeptide”.

Preferably, a polypeptide according to the above-described aspects ofthe invention functions as a metalloprotease. The term “metalloprotease”is well understood in the art and the skilled worker will readily beable to ascertain metalloprotease activity using one of a variety ofassays known in the art. For example, two commonly-applied assays arethe quantitative [³H] gelatin assay (Martin et al., Kidney Int. 36,790-801) and the gelatin zymography assay (Herron G. S. et al., J. Biol.Chem. 1986, 261, 2814-2818).

More preferably, a polypeptide according to the above-described aspectsof the invention is a member of the choriolysin/astacin-like family ofmetalloproteases.

Evidence is presented in the Examples section below that delivery ofINSP005b cDNA (also referred to herein as IPAAA78836-2) in an in vivomodel of fulminant hepatitis was found to decrease TNF-alpha and m-IL-6levels in serum and had a significant effect on the reduction oftransaminases measured in serum.

The decrease in aspartate aminotransferase (ASAT) and alanineaminotransferase (ALAT) levels noted might be due to decreased TNF-alphaand IL-6 levels. TNF-alpha is an important cytokine involved in liverdamage after ConA injection. In this mouse model of liver hepatitis,TNF-alpha is mainly produced by hepatic macrophages, the so-calledKupfer cells. Anti TNF-alpha antibodies have been shown to conferprotection against disease (Seino et al. 2001, Annals of surgery 234,681). Accordingly, it is considered that INSP005b polypeptide andrelated functionally equivalent proteins will be useful in treatingauto-immune, viral or acute liver diseases as well as alcoholic liverfailures. They are likely also to be effective in treating otherinflammatory diseases.

The INSP005a polypeptides, INSP005b polypeptides and the INSP005b maturepolypeptides are referred to herein as “the INSP005 polypeptides”.

In a second aspect, the invention provides a purified nucleic acidmolecule which encodes a polypeptide of the first aspect of theinvention. Preferably, the purified nucleic acid molecule has thenucleic acid sequence as recited in SEQ ID NO:13 (encoding the INSP005apolypeptide), SEQ ID NO:33 (encoding the INSP005b polypeptide) or SEQ IDNO:35 (encoding the INSP005b mature polypeptide), or is a redundantequivalent or fragment of either of these sequences.

In a third aspect, the invention provides a purified nucleic acidmolecule which hydridizes under high stringency conditions with anucleic acid molecule of the second aspect of the invention.

In a fourth aspect, the invention provides a vector, such as anexpression vector, that contains a nucleic acid molecule of the secondor third aspect of the invention. In a preferred embodiment of thisaspect of the invention the vector is the PCR-TOPO-IPAAA78836-1 vector(see FIG. 9 and SEQ ID NO:38). In a further preferred embodiment of thisaspect of the invention the vector is the PCR-TOPO-IPAAA78836-2 vector(see FIG. 12 and SEQ ID NO:39).

In a fifth aspect, the invention provides a host cell transformed with avector of the fourth aspect of the invention.

In a sixth aspect, the invention provides a ligand which bindsspecifically to, and which preferably inhibits the metalloproteaseactivity of a polypeptide of the first aspect of the invention. Ligandsto a polypeptide according to the invention may come in various forms,including natural or modified substrates, enzymes, receptors, smallorganic molecules such as small natural or synthetic organic moleculesof up to 2000 Da, preferably 800 Da or less, peptidomimetics, inorganicmolecules, peptides, polypeptides, antibodies, structural or functionalmimetics of the aforementioned.

In a seventh aspect, the invention provides a compound that is effectiveto alter the expression of a natural gene which encodes a polypeptide ofthe first aspect of the invention or to regulate the activity of apolypeptide of the first aspect of the invention.

A compound of the seventh aspect of the invention may either increase(agonise) or decrease (antagonise) the level of expression of the geneor the activity of the polypeptide. Importantly, the identification ofthe function of the INSP005 polypeptides allows for the design ofscreening methods capable of identifying compounds that are effective inthe treatment and/or diagnosis of disease. Ligands and compoundsaccording to the sixth and seventh aspects of the invention may beidentified using such methods. These methods are included as aspects ofthe present invention.

Evidence is presented in the Examples section below that the INSP005bpolypeptide may be used to prevent or treat inflammatory diseases,auto-immune diseases, liver disease or liver failure. Accordingly, theprovision of a compound according to the seventh aspect of the inventionwhich mimics the INSP005b polypeptide conformationally, or is an agonistof the INSP005b polypeptide is particularly preferred since such acompound may find utility in the prevention or treatment of aninflammatory disease, an auto-immune disease, liver disease or liverfailure as described above.

In an eighth aspect, the invention provides a polypeptide of the firstaspect of the invention, or a nucleic acid molecule of the second orthird aspect of the invention, or a vector of the fourth aspect of theinvention, or a host cell of the fifth aspect of the invention, or aligand of the sixth aspect of the invention, or a compound of theseventh aspect of the invention, for use in therapy or diagnosis ofdiseases in which metalloproteases are implicated. These molecules mayalso be used in the manufacture of a medicament for the treatment ofsuch diseases, particularly respiratory disorders, including emphysemaand cystic fibrosis, metabolic disorders, cardiovascular disorders,bacterial infections, hypertension, proliferative disorders, includingcancer, autoimmune/inflammatory disorders, including rheumatoidarthritis, neurological disorders, developmental disorders andreproductive disorders. These moieties of the first, second, third,fourth, fifth, sixth or seventh aspect of the invention may also be usedin the manufacture of a medicament for the treatment of such diseases.

It is particularly preferred that the moieties of the first, second,third, fourth, fifth and sixth aspects of the invention are used in themanufacture of a medicament for the treatment of inflammatory diseases,autoimmune diseases, liver disease (including viral or acute liverdisease) and liver failure (including alcoholic liver failure).

In a ninth aspect, the invention provides a method of diagnosing adisease in a patient, comprising assessing the level of expression of anatural gene encoding a polypeptide of the first aspect of the inventionor the activity of a polypeptide of the first aspect of the invention intissue from said patient and comparing said level of expression oractivity to a control level, wherein a level that is different to saidcontrol level is indicative of disease. Such a method will preferably becarried out in vitro. Similar methods may be used for monitoring thetherapeutic treatment of disease in a patient, wherein altering thelevel of expression or activity of a polypeptide or nucleic acidmolecule over the period of time towards a control level is indicativeof regression of disease.

A preferred disease diagnosed by a method of the ninth aspect of theinvention is an inflammatory disease, autoimmune disease, liver disease(including viral or acute liver disease) or liver failure (includingalcoholic liver failure).

A preferred method for detecting polypeptides of the first aspect of theinvention comprises the steps of: (a) contacting a ligand, such as anantibody, of the sixth aspect of the invention with a biological sampleunder conditions suitable for the formation of a ligand-polypeptidecomplex; and (b) detecting said complex.

A number of different such methods according to the ninth aspect of theinvention exist, as the skilled reader will be aware, such as methods ofnucleic acid hybridization with short probes, point mutation analysis,polymerase chain reaction (PCR) amplification and methods usingantibodies to detect aberrant protein levels. Similar methods may beused on a short or long term basis to allow therapeutic treatment of adisease to be monitored in a patient. The invention also provides kitsthat are useful in these methods for diagnosing disease.

In a tenth aspect, the invention provides for the use of a polypeptideof the first aspect of the invention as a secreted protein, preferablyas a metalloprotease.

In an eleventh aspect, the invention provides a pharmaceuticalcomposition comprising a polypeptide of the first aspect of theinvention, or a nucleic acid molecule of the second or third aspect ofthe invention, or a vector of the fourth aspect of the invention, or ahost cell of the fifth aspect of the invention, or a ligand of the sixthaspect of the invention, or a compound of the seventh aspect of theinvention, in conjunction with a pharmaceutically-acceptable carrier.

In a twelfth aspect, the present invention provides a polypeptide of thefirst aspect of the invention, or a nucleic acid molecule of the secondor third aspect of the invention, or a vector of the fourth aspect ofthe invention, or a host cell of the fifth aspect of the invention, or aligand of the sixth aspect of the invention, or a compound of theseventh aspect of the invention, for use in the manufacture of amedicament for the diagnosis or treatment of a disease, such asrespiratory disorders, including emphysema and cystic fibrosis,metabolic disorders, cardiovascular disorders, bacterial infection,hypertension, proliferative disorders, including cancer,autoimmune/inflammatory disorders, including rheumatoid arthritis,neurological disorders, developmental disorders, reproductive disordersor other diseases in which metalloproteases are implicated.

It is particularly preferred that the moieties of the first, second,third, fourth, fifth and sixth aspects of the invention are used in themanufacture of a medicament for the treatment of an inflammatorydisease, an auto-immune disease, liver disease or liver failure.

In a thirteenth aspect, the invention provides a method of treating adisease in a patient comprising administering to the patient apolypeptide of the first aspect of the invention, or a nucleic acidmolecule of the second or third aspect of the invention, or a vector ofthe fourth aspect of the invention, or a host cell of the fifth aspectof the invention, or a ligand of the sixth aspect of the invention, or acompound of the seventh aspect of the invention.

For diseases in which the expression of a natural gene encoding apolypeptide of the first aspect of the invention, or in which theactivity of a polypeptide of the first aspect of the invention, is lowerin a diseased patient when compared to the level of expression oractivity in a healthy patient, the polypeptide, nucleic acid molecule,vector, host cell, ligand or compound administered to the patient shouldbe an agonist. Conversely, for diseases in which the expression of thenatural gene or activity of the polypeptide is higher in a diseasedpatient when compared to the level of expression or activity in ahealthy patient, the polypeptide, nucleic acid molecule, vector, hostcell, ligand or compound administered to the patient should be anantagonist. Examples of such antagonists include antisense nucleic acidmolecules, ribozymes and ligands, such as antibodies.

It is particularly preferred that the disease is an inflammatorydisease, an auto-immune disease, liver disease or liver failure.

In a fourteenth aspect, the invention provides transgenic or knockoutnon-human animals that have been transformed to express higher, lower orabsent levels of a polypeptide of the first aspect of the invention.Such transgenic animals are very useful models for the study of diseaseand may also be used in screening regimes for the identification ofcompounds that are effective in the treatment or diagnosis of such adisease.

It is particularly preferred that the disease is an inflammatorydisease, an auto-immune disease, liver disease or liver failure.

A summary of standard techniques and procedures which may be employed inorder to utilise the invention is given below. It will be understoodthat this invention is not limited to the particular methodology,protocols, cell lines, vectors and reagents described. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only and it is not intended that thisterminology should limit the scope of the present invention. The extentof the invention is limited only by the terms of the appended claims.

Standard abbreviations for nucleotides and amino acids are used in thisspecification.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA technology and immunology, which are within the skill ofthose working in the art.

Such techniques are explained fully in the literature. Examples ofparticularly suitable texts for consultation include the following:Sambrook Molecular Cloning; A Laboratory Manual, Second Edition (1989);DNA Cloning, Volumes I and II (D. N Glover ed. 1985); OligonucleotideSynthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames& S. J. Higgins eds. 1984); Transcription and Translation (B. D. Hames &S. J. Higgins eds. 1984); Animal Cell Culture (R. I. Freshney ed. 1986);Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A PracticalGuide to Molecular Cloning (1984); the Methods in Enzymology series(Academic Press, Inc.), especially volumes 154 & 155; Gene TransferVectors for Mammalian Cells (J. H. Miller and M. P. Calos eds. 1987,Cold Spring Harbor Laboratory); Immunochemical Methods in Cell andMolecular Biology (Mayer and Walker, eds. 1987, Academic Press, London);Scopes, (1987) Protein Purification: Principles and Practice, SecondEdition (Springer Verlag, N.Y.); and Handbook of ExperimentalImmunology, Volumes I-IV (D. M. Weir and C. C. Blackwell eds. 1986).

As used herein, the term “polypeptide” includes any peptide or proteincomprising two or more amino acids joined to each other by peptide bondsor modified peptide bonds, i.e. peptide isosteres. This term refers bothto short chains (peptides and oligopeptides) and to longer chains(proteins).

As described above, the polypeptides of the present invention may be inthe form of a mature protein or may be a pre-, pro- or prepro-proteinthat can be activated by cleavage of the pre-, pro- or prepro-portion toproduce an active mature polypeptide. In such polypeptides, the pre-,pro- or prepro-sequence may be a leader or secretory sequence or may bea sequence that is employed for purification of the mature polypeptidesequence.

The polypeptide of the first aspect of the invention may form part of afusion protein. For example, it is often advantageous to include one ormore additional amino acid sequences which may contain secretory orleader sequences, pro-sequences, sequences which aid in purification, orsequences that confer higher protein stability, for example duringrecombinant production. Alternatively or additionally, the maturepolypeptide may be fused with another compound, such as a compound toincrease the half-life of the polypeptide (for example, polyethyleneglycol).

Polypeptides may contain amino acids other than the 20 gene-encodedamino acids, modified either by natural processes, such as bypost-translational processing or by chemical modification techniqueswhich are well known in the art. Among the known modifications which maycommonly be present in polypeptides of the present invention areglycosylation, lipid attachment, sulphation, gamma-carboxylation, forinstance of glutamic acid residues, hydroxylation and ADP-ribosylation.Other potential modifications include acetylation, acylation, amidation,covalent attachment of flavin, covalent attachment of a haeme moiety,covalent attachment of a nucleotide or nucleotide derivative, covalentattachment of a lipid derivative, covalent attachment ofphosphatidylinositol, cross-linking, cyclization, disulphide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation, GPI anchorformation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, transfer-RNA mediated addition of amino acids to proteinssuch as arginylation, and ubiquitination.

Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.In fact, blockage of the amino or carboxyl terminus in a polypeptide, orboth, by a covalent modification is common in naturally-occurring andsynthetic polypeptides and such modifications may be present inpolypeptides of the present invention.

The modifications that occur in a polypeptide often will be a functionof how the polypeptide is made. For polypeptides that are maderecombinantly, the nature and extent of the modifications in large partwill be determined by the post-translational modification capacity ofthe particular host cell and the modification signals that are presentin the amino acid sequence of the polypeptide in question. For instance,glycosylation patterns vary between different types of host cell.

The polypeptides of the present invention can be prepared in anysuitable manner. Such polypeptides include, where the polypeptide is anaturally occurring polypeptide, isolated naturally-occurringpolypeptides (for example purified from cell culture) and alsorecombinantly-produced polypeptides (including fusion proteins),synthetically-produced polypeptides or polypeptides that are produced bya combination of these methods. The term “isolated” does not denote themethod by which the polypeptide is obtained or the level of purity ofthe preparation. Thus, such isolated species may be producedrecombinantly, isolated directly from the cell or tissue of interest orproduced synthetically based on the determined sequences.

The functionally-equivalent polypeptides of the first aspect of theinvention may be polypeptides that are homologous to the INSP005polypeptides. Two polypeptides are said to be “homologous”, as the termis used herein, if the sequence of one of the polypeptides has a highenough degree of identity or similarity to the sequence of the otherpolypeptide. “Identity” indicates that at any particular position in thealigned sequences, the amino acid residue is identical between thesequences. “Similarity” indicates that, at any particular position inthe aligned sequences, the amino acid residue is of a similar typebetween the sequences. Degrees of identity and similarity can be readilycalculated (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing. Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991).

Homologous polypeptides therefore include natural biological variants(for example, allelic variants or geographical variations within thespecies from which the polypeptides are derived) and mutants (such asmutants containing amino acid substitutions, insertions or deletions) ofthe INSP005 polypeptides. Such mutants may include polypeptides in whichone or more of the amino acid residues are substituted with a conservedor non-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code. Typical such substitutions are among Ala,Val, Leu and Ile; among Ser and Thr; among the acidic residues Asp andGlu; among Asn and Gln; among the basic residues Lys and Arg; or amongthe aromatic residues Phe and Tyr. Particularly preferred are variantsin which several, i.e. between 5 and 10, 1 and 5, 1 and 3, 1 and 2 orjust 1 amino acids are substituted, deleted or added in any combination.Especially preferred are silent substitutions, additions and deletions,which do not alter the properties and activities of the protein. Alsoespecially preferred in this regard are conservative substitutions.

Such mutants also include polypeptides in which one or more of the aminoacid residues includes a substituent group;

Typically, greater than 30% identity between two polypeptides isconsidered to be an indication of functional equivalence. Preferably,functionally equivalent polypeptides of the first aspect of theinvention have a degree of sequence identity with the INSP005polypeptides, or with active fragments thereof, of greater than 80%.More preferred polypeptides have degrees of identity of greater than85%, 90%, 95%, 98%, 99% or more, respectively.

The functionally-equivalent polypeptides of the first aspect of theinvention may also be polypeptides which have been identified using oneor more techniques of structural alignment. For example, theInpharmatica Genome Threader technology that forms one aspect of thesearch tools used to generate the Biopendium search database may be used(see PCT patent application WO 01/69507) to identify polypeptides ofpresently-unknown function which, while having low sequence identity ascompared to the INSP005 polypeptides, are predicted to have secretedmolecule activity, by virtue of sharing significant structural homologywith the INSP005 polypeptide sequences. By “significant structuralhomology” is meant that the Inpharmatica Genome Threader predicts twoproteins to share structural homology with a certainty of 10% and above.

The polypeptides of the first aspect of the invention also includefragments of the INSP005 polypeptides and fragments of the functionalequivalents of the INSP005 polypeptides, provided that those fragmentsretain metalloprotease activity or have an antigenic determinant incommon with the INSP005 polypeptides.

As used herein, the term “fragment” refers to a polypeptide having anamino acid sequence that is the same as part, but not all, of the aminoacid sequence of the INSP005 polypeptides or one of its functionalequivalents. The fragments should comprise at least n consecutive aminoacids from the sequence and, depending on the particular sequence, npreferably is 7 or more (for example, 8, 10, 12, 14, 16, 18, 20 ormore). Small fragments may form an antigenic determinant.

Such fragments may be “free-standing”, i.e. not part of or fused toother amino acids or polypeptides, or they may be comprised within alarger polypeptide of which they form a part or region. When comprisedwithin a larger polypeptide, the fragment of the invention mostpreferably forms a single continuous region. For instance, certainpreferred embodiments relate to a fragment having a pre- and/orpro-polypeptide region fused to the amino terminus of the fragmentand/or an additional region fused to the carboxyl terminus of thefragment. However, several fragments may be comprised within a singlelarger polypeptide.

The polypeptides of the present invention or their immunogenic fragments(comprising at least one antigenic determinant) can be used to generateligands, such as polyclonal or monoclonal antibodies, that areimmunospecific for the polypeptides. Such antibodies may be employed toisolate or to identify clones expressing the polypeptides of theinvention or to purify the polypeptides by affinity chromatography. Theantibodies may also be employed as diagnostic or therapeutic aids,amongst other applications, as will be apparent to the skilled reader.

The term “immunospecific” means that the antibodies have substantiallygreater affinity for the polypeptides of the invention than theiraffinity for other related polypeptides in the prior art. As usedherein, the term “antibody” refers to intact molecules as well as tofragments thereof, such as Fab, F(ab′)2 and Fv, which are capable ofbinding to the antigenic determinant in question. Such antibodies thusbind to the polypeptides of the first aspect of the invention.

By “substantially greater affinity” we mean that there is a measurableincrease in the affinity for a polypeptide of the invention as comparedwith the affinity for known secreted proteins.

Preferably, the affinity is at least 1.5-fold, 2-fold, 5-fold 10-fold,100-fold, 10³-fold, 10⁴-fold, 10⁵-fold or 10⁶-fold greater for apolypeptide of the invention than for known secreted proteins.

If polyclonal antibodies are desired, a selected mammal, such as amouse, rabbit, goat or horse, may be immunised with a polypeptide of thefirst aspect of the invention. The polypeptide used to immunise theanimal can be derived by recombinant DNA technology or can besynthesized chemically. If desired, the polypeptide can be conjugated toa carrier protein. Commonly used carriers to which the polypeptides maybe chemically coupled include bovine serum albumin, thyroglobulin andkeyhole limpet haemocyanin. The coupled polypeptide is then used toimmunise the animal. Serum from the immunised animal is collected andtreated according to known procedures, for example by immunoaffinitychromatography.

Monoclonal antibodies to the polypeptides of the first aspect of theinvention can also be readily produced by one skilled in the art. Thegeneral methodology for making monoclonal antibodies using hybridomatechnology is well known (see, for example, Kohler, G. and Milstein, C.,Nature 256: 495-497 (1975); Kozbor et al., Immunology Today 4: 72(1983); Cole et al., 77-96 in Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, Inc. (1985).

Panels of monoclonal antibodies produced against the polypeptides of thefirst aspect of the invention can be screened for various properties,i.e., for isotype, epitope, affinity, etc. Monoclonal antibodies areparticularly useful in purification of the individual polypeptidesagainst which they are directed. Alternatively, genes encoding themonoclonal antibodies of interest may be isolated from hybridomas, forinstance by PCR techniques known in the art, and cloned and expressed inappropriate vectors.

Chimeric antibodies, in which non-human variable regions are joined orfused to human constant regions (see, for example, Liu et al., Proc.Natl. Acad. Sci. USA, 84, 3439 (1987)), may also be of use.

The antibody may be modified to make it less immunogenic in anindividual, for example by humanisation (see Jones et al., Nature, 321,522 (1986); Verhoeyen et al., Science, 239, 1534 (1988); Kabat et al.,J. Immunol., 147, 1709 (1991); Queen et al., Proc. Natl Acad. Sci. USA,86, 10029 (1989); Gorman et al., Proc. Natl Acad. Sci. USA, 88, 34181(1991); and Hodgson et al., Bio/Technology, 9, 421 (1991)). The term“humanised antibody”, as used herein, refers to antibody molecules inwhich the CDR amino acids and selected other amino acids in the variabledomains of the heavy and/or light chains of a non-human donor antibodyhave been substituted in place of the equivalent amino acids in a humanantibody. The humanised antibody thus closely resembles a human antibodybut has the binding ability of the donor antibody.

In a further alternative, the antibody may be a “bispecific” antibody,that is an antibody having two different antigen binding domains, eachdomain being directed against a different epitope.

Phage display technology may be utilised to select genes which encodeantibodies with binding activities towards the polypeptides of theinvention either from repertoires of PCR amplified V-genes oflymphocytes from humans screened for possessing the relevant antibodies,or from naive libraries (McCafferty, J. et al., (1990), Nature 348,552-554; Marks, J. et al., (1992) Biotechnology 10, 779-783). Theaffinity of these antibodies can also be improved by chain shuffling(Clackson, T. et al., (1991) Nature 352, 624-628).

Antibodies generated by the above techniques, whether polyclonal ormonoclonal, have additional utility in that they may be employed asreagents in immunoassays, radioimmunoassays (RIA) or enzyme-linkedimmunosorbent assays (ELISA). In these applications, the antibodies canbe labelled with an analytically-detectable reagent such as aradioisotope, a fluorescent molecule or an enzyme.

Preferred nucleic acid molecules of the second and third aspects of theinvention are those which encode the polypeptide sequences recited inSEQ ID NO:14, SEQ ID NO:34, or SEQ ID NO:36 and functionally equivalentpolypeptides. These nucleic acid molecules may be used in the methodsand applications described herein. The nucleic acid molecules of theinvention preferably comprise at least n consecutive nucleotides fromthe sequences disclosed herein where, depending on the particularsequence, n is 10 or more (for example, 12, 14, 15, 18, 20, 25, 30, 35,40 or more).

The nucleic acid molecules of the invention also include sequences thatare complementary to nucleic acid molecules described above (forexample, for antisense or probing purposes).

Nucleic acid molecules of the present invention may be in the form ofRNA, such as mRNA, or in the form of DNA, including, for instance cDNA,synthetic DNA or genomic DNA. Such nucleic acid molecules may beobtained by cloning, by chemical synthetic techniques or by acombination thereof. The nucleic acid molecules can be prepared, forexample, by chemical synthesis using techniques such as solid phasephosphoramidite chemical synthesis, from genomic or cDNA libraries or byseparation from an organism. RNA molecules may generally be generated bythe in vitro or in vivo transcription of DNA sequences.

The nucleic acid molecules may be double-stranded or single-stranded.Single-stranded DNA may be the coding strand, also known as the sensestrand, or it may be the non-coding strand, also referred to as theanti-sense strand.

The term “nucleic acid molecule” also includes analogues of DNA and RNA,such as those containing modified backbones, and peptide nucleic acids(PNA). The term “PNA”, as used herein, refers to an antisense moleculeor an anti-gene agent which comprises an oligonucleotide of at leastfive nucleotides in length linked to a peptide backbone of amino acidresidues, which preferably ends in lysine. The terminal lysine conferssolubility to the composition. PNAs may be pegylated to extend theirlifespan in a cell, where they preferentially bind complementary singlestranded DNA and RNA and stop transcript elongation (Nielsen, P. E. etal. (1993) Anticancer Drug Des. 8:53-63).

A nucleic acid molecule which encodes the polypeptide of SEQ ID NO:14may be identical to the coding sequence of the nucleic acid moleculeshown in SEQ ID NO:13. A nucleic acid molecule which encodes thepolypeptide of SEQ ID NO:34 may be identical to the coding sequence ofthe nucleic acid molecule shown in SEQ ID NO:33. A nucleic acid moleculewhich encodes the polypeptide of SEQ ID NO:36 may be identical to thecoding sequence of the nucleic acid molecule shown in SEQ ID NO:35.

These molecules also may have a different sequence which, as a result ofthe degeneracy of the genetic code, encodes a polypeptide of SEQ IDNO:14, SEQ ID NO:34 or SEQ ID NO:36. Such nucleic acid molecules mayinclude, but are not limited to, the coding sequence for the maturepolypeptide by itself; the coding sequence for the mature polypeptideand additional coding sequences, such as those encoding a leader orsecretory sequence, such as a pro-, pre- or prepro-polypeptide sequence;the coding sequence of the mature polypeptide, with or without theaforementioned additional coding sequences, together with furtheradditional, non-coding sequences, including non-coding 5′ and 3′sequences, such as the transcribed, non-translated sequences that play arole in transcription (including termination signals), ribosome bindingand mRNA stability. The nucleic acid molecules may also includeadditional sequences which encode additional amino acids, such as thosewhich provide additional functionalities.

The nucleic acid molecules of the second and third aspects of theinvention may also encode the fragments or the functional equivalents ofthe polypeptides and fragments of the first aspect of the invention.Such a nucleic acid molecule may be a naturally-occurring variant suchas a naturally-occurring allelic variant, or the molecule may be avariant that is not known to occur naturally. Such non-naturallyoccurring variants of the nucleic acid molecule may be made bymutagenesis techniques, including those applied to nucleic acidmolecules, cells or organisms.

Among variants in this regard are variants that differ from theaforementioned nucleic acid molecules by nucleotide substitutions,deletions or insertions. The substitutions, deletions or insertions mayinvolve one or more nucleotides. The variants may be altered in codingor non-coding regions or both. Alterations in the coding regions mayproduce conservative or non-conservative amino acid substitutions,deletions or insertions.

The nucleic acid molecules of the invention can also be engineered,using methods generally known in the art, for a variety of reasons,including modifying the cloning, processing, and/or expression of thegene product (the polypeptide). DNA shuffling by random fragmentationand PCR re-assembly of gene fragments and synthetic oligonucleotides areincluded as techniques which may be used to engineer the nucleotidesequences. Site-directed mutagenesis may be used to insert newrestriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, introduce mutations and so forth.

Nucleic acid molecules which encode a polypeptide of the first aspect ofthe invention may be ligated to a heterologous sequence so that thecombined nucleic acid molecule encodes a fusion protein. Such combinednucleic acid molecules are included within the second or third aspectsof the invention. For example, to screen peptide libraries forinhibitors of the activity of the polypeptide, it may be useful toexpress, using such a combined nucleic acid molecule, a fusion proteinthat can be recognised by a commercially-available antibody. A fusionprotein may also be engineered to contain a cleavage site locatedbetween the sequence of the polypeptide of the invention and thesequence of a heterologous protein so that the polypeptide may becleaved and purified away from the heterologous protein.

The nucleic acid molecules of the invention also include antisensemolecules that are partially complementary to nucleic acid moleculesencoding polypeptides of the present invention and that thereforehybridize to the encoding nucleic acid molecules (hybridization). Suchantisense molecules, such as oligonucleotides, can be designed torecognise, specifically bind to and prevent transcription of a targetnucleic acid encoding a polypeptide of the invention, as will be knownby those of ordinary skill in the art (see, for example, Cohen, J. S.,Trends in Pharm. Sci., 10, 435 (1989), Okano, J. Neurochem. 56, 560(1991); O'Connor, J. Neurochem 56, 560 (1991); Lee et al., Nucleic AcidsRes 6, 3073 (1979); Cooney et al., Science 241, 456 (1988); Dervan etal., Science 251, 1360 (1991).

The term “hybridization” as used here refers to the association of twonucleic acid molecules with one another by hydrogen bonding. Typically,one molecule will be fixed to a solid support and the other will be freein solution. Then, the two molecules may be placed in contact with oneanother under conditions that favour hydrogen bonding. Factors thataffect this bonding include: the type and volume of solvent; reactiontemperature; time of hybridization; agitation; agents to block thenon-specific attachment of the liquid phase molecule to the solidsupport (Denhardt's reagent or BLOTTO); the concentration of themolecules; use of compounds to increase the rate of association ofmolecules (dextran sulphate or polyethylene glycol); and the stringencyof the washing conditions following hybridization (see Sambrook et al.[supra]).

The inhibition of hybridization of a completely complementary moleculeto a target molecule may be examined using a hybridization assay, asknown in the art (see, for example, Sambrook et al [supra]). Asubstantially homologous molecule will then compete for and inhibit thebinding of a completely homologous molecule to the target molecule undervarious conditions of stringency, as taught in Wahl, G. M. and S. L.Berger (1987; Methods Enzymol. 152:399-407) and Kimmel, A. R. (1987;Methods Enzymol. 152:507-511).

“Stringency” refers to conditions in a hybridization reaction thatfavour the association of very similar molecules over association ofmolecules that differ. High stringency hybridisation conditions aredefined as overnight incubation at 42° C in a solution comprising 50%formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodiumphosphate (pH 7.6), 5× Denhardts solution, 10% dextran sulphate, and 20microgram/ml denatured, sheared salmon sperm DNA, followed by washingthe filters in 0.1×SSC at approximately 65° C. Low stringency conditionsinvolve the hybridisation reaction being carried out at 35° C (seeSambrook et al. [supra]). Preferably, the conditions used forhybridization are those of high stringency.

Preferred embodiments of this aspect of the invention are nucleic acidmolecules that are at least 70% identical over their entire length tonucleic acid molecules encoding the INSP005 polypeptides (SEQ ID NO:13,SEQ ID NO:33 and SEQ ID NO:35), and nucleic acid molecules that aresubstantially complementary to such nucleic acid molecules. Preferably,a nucleic acid molecule according to this aspect of the inventioncomprises a region that is at least 80% identical over its entire lengthto the nucleic acid molecule having the sequence given in SEQ ID NO:13,SEQ ID NO:33 or SEQ ID NO:35 or a nucleic acid molecule that iscomplementary thereto. In this regard, nucleic acid molecules at least90%, preferably at least 95%, more preferably at least 98% or 99%identical over their entire length to the same are particularlypreferred. Preferred embodiments in this respect are nucleic acidmolecules that encode polypeptides which retain substantially the samebiological function or activity as the INSP005 polypeptides.

The invention also provides a process for detecting a nucleic acidmolecule of the invention, comprising the steps of: (a) contacting anucleic probe according to the invention with a biological sample underhybridizing conditions to form duplexes; and (b) detecting any suchduplexes that are formed.

As discussed additionally below in connection with assays that may beutilised according to the invention, a nucleic acid molecule asdescribed above may be used as a hybridization probe for RNA, cDNA orgenomic DNA, in order to isolate full-length cDNAs and genomic clonesencoding the INSP005 polypeptides and to isolate cDNA and genomic clonesof homologous or orthologous genes that have a high sequence similarityto the gene encoding these polypeptides.

In this regard, the following techniques, among others known in the art,may be utilised and are discussed below for purposes of illustration.Methods for DNA sequencing and analysis are well known and are generallyavailable in the art and may, indeed, be used to practice many of theembodiments of the invention discussed herein. Such methods may employsuch enzymes as the Klenow fragment of DNA polymerase 1, Sequenase (USBiochemical Corp, Cleveland, Ohio), Taq polymerase (Perkin Elmer),thermostable T7 polymerase (Amersham, Chicago, Ill.), or combinations ofpolymerases and proof-reading exonucleases such as those found in theELONGASE Amplification System marketed by Gibco/BRL (Gaithersburg, Md.).Preferably, the sequencing process may be automated using machines suchas the Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.), the PeltierThermal Cycler (PTC200; MJ Research, Watertown, Mass.) and the ABICatalyst and 373 and 377 DNA Sequencers (Perkin Elmer).

One method for isolating a nucleic acid molecule encoding a polypeptidewith an equivalent function to that of the INSP005 polypeptides is toprobe a genomic or cDNA library with a natural or artificially-designedprobe using standard procedures that are recognised in the art (see, forexample, “Current Protocols in Molecular Biology”, Ausubel et al. (eds).Greene Publishing Association and John Wiley Interscience, New York,1989, 1992). Probes comprising at least 15, preferably at least 30, andmore preferably at least 50, contiguous bases that correspond to, or arecomplementary to, nucleic acid sequences from the appropriate encodinggene (SEQ ID NO:13, SEQ ID NO:33 or SEQ ID NO:35), are particularlyuseful probes. Such probes may be labelled with ananalytically-detectable reagent to facilitate their identification.Useful reagents include, but are not limited to, radioisotopes,fluorescent dyes and enzymes that are capable of catalysing theformation of a detectable product. Using these probes, the ordinarilyskilled artisan will be capable of isolating complementary copies ofgenomic DNA, cDNA or RNA polynucleotides encoding proteins of interestfrom human, mammalian or other animal sources and screening such sourcesfor related sequences, for example, for additional members of thefamily, type and/or subtype.

In many cases, isolated cDNA sequences will be incomplete, in that theregion encoding the polypeptide will be cut short, normally at the 5′end. Several methods are available to obtain full length cDNAs, or toextend short cDNAs. Such sequences may be extended utilising a partialnucleotide sequence and employing various methods known in the art todetect upstream sequences such as promoters and regulatory elements. Forexample, one method which may be employed is based on the method ofRapid Amplification of cDNA Ends (RACE; see, for example, Frohman etal., PNAS USA 85, 8998-9002, 1988). Recent modifications of thistechnique, exemplified by the Marathon™ technology (ClontechLaboratories Inc.), for example, have significantly simplified thesearch for longer cDNAs. A slightly different technique, termed“restriction-site” PCR, uses universal primers to retrieve unknownnucleic acid sequence adjacent a known locus (Sarkar, G. (1993) PCRMethods Applic. 2:318-322). Inverse PCR may also be used to amplify orto extend sequences using divergent primers based on a known region(Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186). Another methodwhich may be used is capture PCR which involves PCR amplification of DNAfragments adjacent a known sequence in human and yeast artificialchromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic., 1,111-119). Another method which may be used to retrieve unknown sequencesis that of Parker, J. D. et al. (1991); Nucleic Acids Res.19:3055-3060). Additionally, one may use PCR, nested primers, andPromoterFinder™ libraries to walk genomic DNA (Clontech, Palo Alto,Calif.). This process avoids the need to screen libraries and is usefulin finding intron/exon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences that contain the 5′ regions of genes. Use of a randomly primedlibrary may be especially preferable for situations in which an oligod(T) library does not yield a full-length cDNA. Genomic libraries may beuseful for extension of sequence into 5′ non-transcribed regulatoryregions.

In one embodiment of the invention, the nucleic acid molecules of thepresent invention may be used for chromosome localisation. In thistechnique, a nucleic acid molecule is specifically targeted to, and canhybridize with, a particular location on an individual human chromosome.The mapping of relevant sequences to chromosomes according to thepresent invention is an important step in the confirmatory correlationof those sequences with the gene-associated disease. Once a sequence hasbeen mapped to a precise chromosomal location, the physical position ofthe sequence on the chromosome can be correlated with genetic map data.Such data are found in, for example, V. McKusick, Mendelian Inheritancein Man (available on-line through Johns Hopkins University Welch MedicalLibrary). The relationships between genes and diseases that have beenmapped to the same chromosomal region are then identified throughlinkage analysis (coinheritance of physically adjacent genes). Thisprovides valuable information to investigators searching for diseasegenes using positional cloning or other gene discovery techniques. Oncethe disease or syndrome has been crudely localised by genetic linkage toa particular genomic region, any sequences mapping to that area mayrepresent associated or regulatory genes for further investigation. Thenucleic acid molecule may also be used to detect differences in thechromosomal location due to translocation, inversion, etc. among normal,carrier, or affected individuals.

The nucleic acid molecules of the present invention are also valuablefor tissue localisation. Such techniques allow the determination ofexpression patterns of the polypeptide in tissues by detection of themRNAs that encode them. These techniques include in situ hybridizationtechniques and nucleotide amplification techniques, such as PCR. Resultsfrom these studies provide an indication of the normal functions of thepolypeptide in the organism. In addition, comparative studies of thenormal expression pattern of mRNAs with that of mRNAs encoded by amutant gene provide valuable insights into the role of mutantpolypeptides in disease. Such inappropriate expression may be of atemporal, spatial or quantitative nature.

Gene silencing approaches may also be undertaken to down-regulateendogenous expression of a gene encoding a polypeptide of the invention.RNA interference (RNAi) (Elbashir, S M et al., Nature 2001, 411,494-498) is one method of sequence specific post-transcriptional genesilencing that may be employed. Short dsRNA oligonucleotides aresynthesised in vitro and introduced into a cell. The sequence specificbinding of these dsRNA oligonucleotides triggers the degradation oftarget mRNA, reducing or ablating target protein expression.

Efficacy of the gene silencing approaches assessed above may be assessedthrough the measurement of polypeptide expression (for example, byWestern blotting), and at the RNA level using TaqMan-basedmethodologies.

The vectors of the present invention comprise nucleic acid molecules ofthe invention and may be cloning or expression vectors. The host cellsof the invention, which may be transformed, transfected or transducedwith the vectors of the invention may be prokaryotic or eukaryotic.

The polypeptides of the invention may be prepared in recombinant form byexpression of their encoding nucleic acid molecules in vectors containedwithin a host cell. Such expression methods are well known to those ofskill in the art and many are described in detail by Sambrook et al(supra) and Fernandez & Hoeffler (1998, eds. “Gene expression systems.Using nature for the art of expression”. Academic Press, San Diego,London, Boston, New York, Sydney, Tokyo, Toronto).

Generally, any system or vector that is suitable to maintain, propagateor express nucleic acid molecules to produce a polypeptide in therequired host may be used. The appropriate nucleotide sequence may beinserted into an expression system by any of a variety of well-known androutine techniques, such as, for example, those described in Sambrook etal., (supra). Generally, the encoding gene can be placed under thecontrol of a control element such as a promoter, ribosome binding site(for bacterial expression) and, optionally, an operator, so that the DNAsequence encoding the desired polypeptide is transcribed into RNA in thetransformed host cell.

Examples of suitable expression systems include, for example,chromosomal, episomal and virus-derived systems, including, for example,vectors derived from: bacterial plasmids, bacteriophage, transposons,yeast episomes, insertion elements, yeast chromosomal elements, virusessuch as baculoviruses, papova viruses such as SV40, vaccinia viruses,adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses,or combinations thereof, such as those derived from plasmid andbacteriophage genetic elements, including cosmids and phagemids. Humanartificial chromosomes (HACs) may also be employed to deliver largerfragments of DNA than can be contained and expressed in a plasmid.

Particularly suitable expression systems include microorganisms such asbacteria transformed with recombinant bacteriophage, plasmid or cosmidDNA expression vectors; yeast transformed with yeast expression vectors;insect cell systems infected with virus expression vectors (for example,baculovirus); plant cell systems transformed with virus expressionvectors (for example, cauliflower mosaic virus, CaMV; tobacco mosaicvirus, TMV) or with bacterial expression vectors (for example, Ti orpBR322 plasmids); or animal cell systems. Cell-free translation systemscan also be employed to produce the polypeptides of the invention.

Introduction of nucleic acid molecules encoding a polypeptide of thepresent invention into host cells can be effected by methods describedin many standard laboratory manuals, such as Davis et al., Basic Methodsin Molecular Biology (1986) and Sambrook et al., (supra). Particularlysuitable methods include calcium phosphate transfection, DEAE-dextranmediated transfection, transvection, microinjection, cationiclipid-mediated transfection, electroporation, transduction, scrapeloading, ballistic introduction or infection (see Sambrook et al., 1989[supra]; Ausubel et al., 1991 [supra]; Spector, Goldman & Leinwald,1998). In eukaryotic cells, expression systems may either be transient(for example, episomal) or permanent (chromosomal integration) accordingto the needs of the system.

The encoding nucleic acid molecule may or may not include a sequenceencoding a control sequence, such as a signal peptide or leadersequence, as desired, for example, for secretion of the translatedpolypeptide into the lumen of the endoplasmic reticulum, into theperiplasmic space or into the extracellular environment. These signalsmay be endogenous to the polypeptide or they may be heterologoussignals. Leader sequences can be removed by the bacterial host inpost-translational processing.

In addition to control sequences, it may be desirable to add regulatorysequences that allow for regulation of the expression of the polypeptiderelative to the growth of the host cell. Examples of regulatorysequences are those which cause the expression of a gene to be increasedor decreased in response to a chemical or physical stimulus, includingthe presence of a regulatory compound or to various temperature ormetabolic conditions. Regulatory sequences are those non-translatedregions of the vector, such as enhancers, promoters and 5′ and 3′untranslated regions. These interact with host cellular proteins tocarry out transcription and translation. Such regulatory sequences mayvary in their strength and specificity. Depending on the vector systemand host utilised, any number of suitable transcription and translationelements, including constitutive and inducible promoters, may be used.For example, when cloning in bacterial systems, inducible promoters suchas the hybrid lacZ promoter of the Bluescript phagemid (Stratagene, LaJolla, Calif.) or pSport1™ plasmid (Gibco BRL) and the like may be used.The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (forexample, heat shock, RUBISCO and storage protein genes) or from plantviruses (for example, viral promoters or leader sequences) may be clonedinto the vector. In mammalian cell systems, promoters from mammaliangenes or from mammalian viruses are preferable. If it is necessary togenerate a cell line that contains multiple copies of the sequence,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

An expression vector is constructed so that the particular nucleic acidcoding sequence is located in the vector with the appropriate regulatorysequences, the positioning and orientation of the coding sequence withrespect to the regulatory sequences being such that the coding sequenceis transcribed under the “control” of the regulatory sequences, i.e.,RNA polymerase which binds to the DNA molecule at the control sequencestranscribes the coding sequence. In some cases it may be necessary tomodify the sequence so that it may be attached to the control sequenceswith the appropriate orientation; i.e., to maintain the reading frame.

The control sequences and other regulatory sequences may be ligated tothe nucleic acid coding sequence prior to insertion into a vector.Alternatively, the coding sequence can be cloned directly into anexpression vector that already contains the control sequences and anappropriate restriction site.

For long-term, high-yield production of a recombinant polypeptide,stable expression is preferred. For example, cell lines which stablyexpress the polypeptide of interest may be transformed using expressionvectors which may contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate vector. Following the introduction of the vector, cells may beallowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells that successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type.

Mammalian cell lines available as hosts for expression are known in theart and include many immortalised cell lines available from the AmericanType Culture Collection (ATCC) including, but not limited to, Chinesehamster ovary (CHO), HeLa, baby hamster kidney (BHK), monkey kidney(COS), C127, 3T3, BHK, HEK 293, Bowes melanoma and human hepatocellularcarcinoma (for example Hep G2) cells and a number of other cell lines.

In the baculovirus system, the materials for baculovirus/insect cellexpression systems are commercially available in kit form from, interalia, Invitrogen, San Diego Calif. (the “MaxBac” kit). These techniquesare generally known to those skilled in the art and are described fullyin Summers and Smith, Texas Agricultural Experiment Station Bulletin No.1555 (1987). Particularly suitable host cells for use in this systeminclude insect cells such as Drosophila S2 and Spodoptera Sf9 cells.

There are many plant cell culture and whole plant genetic expressionsystems known in the art. Examples of suitable plant cellular geneticexpression systems include those described in U.S. Pat. Nos. 5,693,506;5,659,122; and 5,608,143. Additional examples of genetic expression inplant cell culture has been described by Zenk, Phytochemistry 30,3861-3863 (1991).

In particular, all plants from which protoplasts can be isolated andcultured to give whole regenerated plants can be utilised, so that wholeplants are recovered which contain the transferred gene. Practically allplants can be regenerated from cultured cells or tissues, including butnot limited to all major species of sugar cane, sugar beet, cotton,fruit and other trees, legumes and vegetables.

Examples of particularly preferred bacterial host cells includestreptococci, staphylococci, E. coli, Streptomyces and Bacillus subtiliscells.

Examples of particularly suitable host cells for fungal expressioninclude yeast cells (for example, S. cerevisiae) and Aspergillus cells.

Any number of selection systems are known in the art that may be used torecover transformed cell lines. Examples include the herpes simplexvirus thymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) andadenine phosphoribosyltransferase (Lowy, I. et al. (1980) Cell22:817-23) genes that can be employed in tk− or aprt± cells,respectively.

Also, antimetabolite, antibiotic or herbicide resistance can be used asthe basis for selection; for example, dihydrofolate reductase (DHFR)that confers resistance to methotrexate (Wigler, M. et al. (1980) Proc.Natl. Acad. Sci. 77:3567-70); npt, which confers resistance to theaminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al (1981) J.Mol. Biol. 150:1-14) and als or pat, which confer resistance tochlorsulfuron and phosphinotricin acetyltransferase, respectively.Additional selectable genes have been described, examples of which willbe clear to those of skill in the art.

Although the presence or absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the relevant sequence is insertedwithin a marker gene sequence, transformed cells containing theappropriate sequences can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding a polypeptide of the invention under the control of asingle promoter. Expression of the marker gene in response to inductionor selection usually indicates expression of the tandem gene as well.

Alternatively, host cells that contain a nucleic acid sequence encodinga polypeptide of the invention and which express said polypeptide may beidentified by a variety of procedures known to those of skill in theart. These procedures include, but are not limited to, DNA-DNA orDNA-RNA hybridizations and protein bioassays, for example, fluorescenceactivated cell sorting (FACS) or immunoassay techniques (such as theenzyme-linked immunosorbent assay [ELISA] and radioimmunoassay [RIA]),that include membrane, solution, or chip based technologies for thedetection and/or quantification of nucleic acid or protein (see Hampton,R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983) J. Exp. Med, 158,1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labelled hybridization or PCR probesfor detecting sequences related to nucleic acid molecules encodingpolypeptides of the present invention include oligolabelling, nicktranslation, end-labelling or PCR amplification using a labelledpolynucleotide. Alternatively, the sequences encoding the polypeptide ofthe invention may be cloned into a vector for the production of an mRNAprobe. Such vectors are known in the art, are commercially available,and may be used to synthesise RNA probes in vitro by addition of anappropriate RNA polymerase such as T7, T3 or SP6 and labellednucleotides. These procedures may be conducted using a variety ofcommercially available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.);Promega (Madison Wis.); and U.S. Biochemical Corp., Cleveland, Ohio)).

Suitable reporter molecules or labels, which may be used for ease ofdetection, include radionuclides, enzymes and fluorescent,chemiluminescent or chromogenic agents as well as substrates, cofactors,inhibitors, magnetic particles, and the like.

Nucleic acid molecules according to the present invention may also beused to create transgenic animals, particularly rodent animals. Suchtransgenic animals form a further aspect of the present invention. Thismay be done locally by modification of somatic cells, or by germ linetherapy to incorporate heritable modifications. Such transgenic animalsmay be particularly useful in the generation of animal models for drugmolecules effective as modulators of the polypeptides of the presentinvention.

The polypeptide can be recovered and purified from recombinant cellcultures by well-known methods including ammonium sulphate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. High performance liquid chromatography is particularlyuseful for purification. Well known techniques for refolding proteinsmay be employed to regenerate an active conformation when thepolypeptide is denatured during isolation and or purification.

Specialised vector constructions may also be used to facilitatepurification of proteins, as desired, by joining sequences encoding thepolypeptides of the invention to a nucleotide sequence encoding apolypeptide domain that will facilitate purification of solubleproteins. Examples of such purification-facilitating domains includemetal chelating peptides such as histidine-tryptophan modules that allowpurification on immobilised metals, protein A domains that allowpurification on immobilised immunoglobulin, and the domain utilised inthe FLAGS extension/affinity purification system (Immunex Corp.,Seattle, Wash.). The inclusion of cleavable linker sequences such asthose specific for Factor XA or enterokinase (Invitrogen, San Diego,Calif.) between the purification domain and the polypeptide of theinvention may be used to facilitate purification. One such expressionvector provides for expression of a fusion protein containing thepolypeptide of the invention fused to several histidine residuespreceding a thioredoxin or an enterokinase cleavage site. The histidineresidues facilitate purification by IMAC (immobilised metal ion affinitychromatography as described in Porath, J. et al. (1992), Prot. Exp.Purif. 3: 263-281) while the thioredoxin or enterokinase cleavage siteprovides a means for purifying the polypeptide from the fusion protein.A discussion of vectors which contain fusion proteins is provided inKroll, D. J. et al. (1993; DNA Cell Biol. 12:441-453).

If the polypeptide is to be expressed for use in screening assays,generally it is preferred that it be produced at the surface of the hostcell in which it is expressed. In this event, the host cells may beharvested prior to use in the screening assay, for example usingtechniques such as fluorescence activated cell sorting (FACS) orimmunoaffinity techniques. If the polypeptide is secreted into themedium, the medium can be recovered in order to recover and purify theexpressed polypeptide. If polypeptide is produced intracellularly, thecells must first be lysed before the polypeptide is recovered.

The polypeptide of the invention can be used to screen libraries ofcompounds in any of a variety of drug screening techniques. Suchcompounds may activate (agonise) or inhibit (antagonise) the level ofexpression of the gene or the activity of the polypeptide of theinvention and form a further aspect of the present invention. Preferredcompounds are effective to alter the expression of a natural gene whichencodes a polypeptide of the first aspect of the invention or toregulate the activity of a polypeptide of the first aspect of theinvention.

Agonist or antagonist compounds may be isolated from, for example,cells, cell-free preparations, chemical libraries or natural productmixtures. These agonists or antagonists may be natural or modifiedsubstrates, ligands, enzymes, receptors or structural or functionalmimetics. For a suitable review of such screening techniques, seeColigan et al., Current Protocols in Immunology 1(2): Chapter 5 (1991).

Compounds that are most likely to be good antagonists are molecules thatbind to the polypeptide of the invention without inducing the biologicaleffects of the polypeptide upon binding to it. Potential antagonistsinclude small organic molecules, peptides, polypeptides and antibodiesthat bind to the polypeptide of the invention and thereby inhibit orextinguish its activity. In this fashion, binding of the polypeptide tonormal cellular binding molecules may be inhibited, such that the normalbiological activity of the polypeptide is prevented.

The polypeptide of the invention that is employed in such a screeningtechnique may be free in solution, affixed to a solid support, borne ona cell surface or located intracellularly. In general, such screeningprocedures may involve using appropriate cells or cell membranes thatexpress the polypeptide that are contacted with a test compound toobserve binding, or stimulation or inhibition of a functional response.The functional response of the cells contacted with the test compound isthen compared with control cells that were not contacted with the testcompound. Such an assay may assess whether the test compound results ina signal generated by activation of the polypeptide, using anappropriate detection system. Inhibitors of activation are generallyassayed in the presence of a known agonist and the effect on activationby the agonist in the presence of the test compound is observed.

The INSP005 polypeptides of the present invention may modulate a varietyof physiological and pathological processes, including reproductiveprocesses such as egg maturation or fertilisation. Thus, the biologicalactivity of the INSP005 polypeptides can be examined in systems thatallow the study of such modulatory activities, using a variety ofsuitable assays. For example, possible assays include the measurement ofoocyte fertilisation and/or pregnancy rates after ovulation induction,the measurement of embryo implantation rates, or in the case of maleinfertility the measurement of sperm motility (Luo C. W. et al, J. Biol.Chem. 276 (10), 6913-6921 (2001)).

A preferred method for identifying an agonist or antagonist compound ofa polypeptide of the present invention comprises:

-   -   (a) contacting a cell expressing on the surface thereof the        polypeptide according to the first aspect of the invention, the        polypeptide being associated with a second component capable of        providing a detectable signal in response to the binding of a        compound to the polypeptide, with a compound to be screened        under conditions to permit binding to the polypeptide; and    -   (b) determining whether the compound binds to and activates or        inhibits the polypeptide by measuring the level of a signal        generated from the interaction of the compound with the        polypeptide.

A further preferred method for identifying an agonist or antagonist of apolypeptide of the invention comprises:

-   -   (a) contacting a cell expressing on the surface thereof the        polypeptide, the polypeptide being associated with a second        component capable of providing a detectable signal in response        to the binding of a compound to the polypeptide, with a compound        to be screened under conditions to permit binding to the        polypeptide; and    -   (b) determining whether the compound binds to and activates or        inhibits the polypeptide by comparing the level of a signal        generated from the interaction of the compound with the        polypeptide with the level of a signal in the absence of the        compound.

In further preferred embodiments, the general methods that are describedabove may further comprise conducting the identification of agonist orantagonist in the presence of labelled or unlabelled ligand for thepolypeptide.

In another embodiment of the method for identifying agonist orantagonist of a polypeptide of the present invention comprises:

determining the inhibition of binding of a ligand to cells which have apolypeptide of the invention on the surface thereof, or to cellmembranes containing such a polypeptide, in the presence of a candidatecompound under conditions to permit binding to the polypeptide, anddetermining the amount of ligand bound to the polypeptide. A compoundcapable of causing reduction of binding of a ligand is considered to bean agonist or antagonist. Preferably the ligand is labelled.

More particularly, a method of screening for a polypeptide antagonist oragonist compound comprises the steps of:

-   -   (a) incubating a labelled ligand with a whole cell expressing a        polypeptide according to the invention on the cell surface, or a        cell membrane containing a polypeptide of the invention,    -   (b) measuring the amount of labelled ligand bound to the whole        cell or the cell membrane;    -   (c) adding a candidate compound to a mixture of labelled ligand        and the whole cell or the cell membrane of step (a) and allowing        the mixture to attain equilibrium;    -   (d) measuring the amount of labelled ligand bound to the whole        cell or the cell membrane after step (c); and    -   (e) comparing the difference in the labelled ligand bound in        step (b) and (d), such that the compound which causes the        reduction in binding in step (d) is considered to be an agonist        or antagonist.

The polypeptides may be found to modulate a variety of physiological andpathological processes in a dose-dependent manner in the above-describedassays. Thus, the “functional equivalents” of the polypeptides of theinvention include polypeptides that exhibit any of the same modulatoryactivities in the above-described assays in a dose-dependent manner.Although the degree of dose-dependent activity need not be identical tothat of the polypeptides of the invention, preferably the “functionalequivalents” will exhibit substantially similar dose-dependence in agiven activity assay compared to the polypeptides of the invention.

In certain of the embodiments described above, simple binding assays maybe used, in which the adherence of a test compound to a surface bearingthe polypeptide is detected by means of a label directly or indirectlyassociated with the test compound or in an assay involving competitionwith a labelled competitor. In another embodiment, competitive drugscreening assays may be used, in which neutralising antibodies that arecapable of binding the polypeptide specifically compete with a testcompound for binding. In this manner, the antibodies can be used todetect the presence of any test compound that possesses specific bindingaffinity for the polypeptide.

Assays may also be designed to detect the effect of added test compoundson the production of mRNA encoding the polypeptide in cells. Forexample, an ELISA may be constructed that measures secreted orcell-associated levels of polypeptide using monoclonal or polyclonalantibodies by standard methods known in the art, and this can be used tosearch for compounds that may inhibit or enhance the production of thepolypeptide from suitably manipulated cells or tissues. The formation ofbinding complexes between the polypeptide and the compound being testedmay then be measured.

Assay methods that are also included within the terms of the presentinvention are those that involve the use of the genes and polypeptidesof the invention in overexpression or ablation assays. Such assaysinvolve the manipulation of levels of these genes/polypeptides in cellsand assessment of the impact of this manipulation event on thephysiology of the manipulated cells. For example, such experimentsreveal details of signaling and metabolic pathways in which theparticular genes/polypeptides are implicated, generate informationregarding the identities of polypeptides with which the studiedpolypeptides interact and provide clues as to methods by which relatedgenes and proteins are regulated.

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe polypeptide of interest (see International patent applicationWO84/03564). In this method, large numbers of different small testcompounds are synthesised on a solid substrate, which may then bereacted with the polypeptide of the invention and washed. One way ofimmobilising the polypeptide is to use non-neutralising antibodies.Bound polypeptide may then be detected using methods that are well knownin the art. Purified polypeptide can also be coated directly onto platesfor use in the aforementioned drug screening techniques.

The polypeptide of the invention may be used to identify membrane-boundor soluble receptors, through standard receptor binding techniques thatare known in the art, such as ligand binding and crosslinking assays inwhich the polypeptide is labelled with a radioactive isotope, ischemically modified, or is fused to a peptide sequence that facilitatesits detection or purification, and incubated with a source of theputative receptor (for example, a composition of cells, cell membranes,cell supernatants, tissue extracts, or bodily fluids). The efficacy ofbinding may be measured using biophysical techniques such as surfaceplasmon resonance (supplied by Biacore AB, Uppsala, Sweden) andspectroscopy. Binding assays may be used for the purification andcloning of the receptor, but may also identify agonists and antagonistsof the polypeptide, that compete with the binding of the polypeptide toits receptor. Standard methods for conducting screening assays are wellunderstood in the art.

The invention also includes a screening kit useful in the methods foridentifying agonists, antagonists, ligands, receptors, substrates,enzymes, that are described above.

The invention includes the agonists, antagonists, ligands, receptors,substrates and enzymes, and other compounds which modulate the activityor antigenicity of the polypeptide of the invention discovered by themethods that are described above.

The invention also provides pharmaceutical compositions comprising apolypeptide, nucleic acid, ligand or compound of the invention incombination with a suitable pharmaceutical carrier. These compositionsmay be suitable as therapeutic or diagnostic reagents, as vaccines, oras other immunogenic compositions, as outlined in detail below.

According to the terminology used herein, a composition containing apolypeptide, nucleic acid, ligand or compound [X] is “substantially freeof” impurities [herein, Y] when at least 85% by weight of the total X+Yin the composition is X. Preferably, X comprises at least about 90% byweight of the total of X+Y in the composition, more preferably at leastabout 95%, 98% or even 99% by weight.

The pharmaceutical compositions should preferably comprise atherapeutically effective amount of the polypeptide, nucleic acidmolecule, ligand, or compound of the invention. The term“therapeutically effective amount” as used herein refers to an amount ofa therapeutic agent needed to treat, ameliorate, or prevent a targeteddisease or condition, or to exhibit a detectable therapeutic orpreventative effect. For any compound, the therapeutically effectivedose can be estimated initially either in cell culture assays, forexample, of neoplastic cells, or in animal models, usually mice,rabbits, dogs, or pigs. The animal model may also be used to determinethe appropriate concentration range and route of administration. Suchinformation can then be used to determine useful doses and routes foradministration in humans.

The precise effective amount for a human subject will depend upon theseverity of the disease state, general health of the subject, age,weight, and gender of the subject, diet, time and frequency ofadministration, drug combination(s), reaction sensitivities, andtolerance/response to therapy. This amount can be determined by routineexperimentation and is within the judgement of the clinician. Generally,an effective dose will be from 0.01 mg/kg to 50 mg/kg, preferably 0.05mg/kg to 10 mg/kg. Compositions may be administered individually to apatient or may be administered in combination with other agents, drugsor hormones.

A pharmaceutical composition may also contain a pharmaceuticallyacceptable carrier, for administration of a therapeutic agent. Suchcarriers include antibodies and other polypeptides, genes and othertherapeutic agents such as liposomes, provided that the carrier does notitself induce the production of antibodies harmful to the individualreceiving the composition, and which may be administered without unduetoxicity. Suitable carriers may be large, slowly metabolisedmacromolecules such as proteins, polysaccharides, polylactic acids,polyglycolic acids, polymeric amino acids, amino acid copolymers andinactive virus particles.

Pharmaceutically acceptable salts can be used therein, for example,mineral acid salts such as hydrochlorides, hydrobromides, phosphates,sulphates, and the like; and the salts of organic acids such asacetates, propionates, malonates, benzoates, and the like. A thoroughdiscussion of pharmaceutically acceptable carriers is available inRemington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

Pharmaceutically acceptable carriers in therapeutic compositions mayadditionally contain liquids such as water, saline, glycerol andethanol. Additionally, auxiliary substances, such as wetting oremulsifying agents, pH buffering substances, and the like, may bepresent in such compositions. Such carriers enable the pharmaceuticalcompositions to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions, and the like, foringestion by the patient.

Once formulated, the compositions of the invention can be administereddirectly to the subject. The subjects to be treated can be animals; inparticular, human subjects can be treated.

The pharmaceutical compositions utilised in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal or transcutaneousapplications (for example, see WO98/20734), subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, intravaginalor rectal means. Gene guns or hyposprays may also be used to administerthe pharmaceutical compositions of the invention. Typically, thetherapeutic compositions may be prepared as injectables, either asliquid solutions or suspensions; solid forms suitable for solution in,or suspension in, liquid vehicles prior to injection may also beprepared.

Direct delivery of the compositions will generally be accomplished byinjection, subcutaneously, intraperitoneally, intravenously orintramuscularly, or delivered to the interstitial space of a tissue. Thecompositions can also be administered into a lesion. Dosage treatmentmay be a single dose schedule or a multiple dose schedule.

If the activity of the polypeptide of the invention is in excess in aparticular disease state, several approaches are available. One approachcomprises administering to a subject an inhibitor compound (antagonist)as described above, along with a pharmaceutically acceptable carrier inan amount effective to inhibit the function of the polypeptide, such asby blocking the binding of ligands, substrates, enzymes, receptors, orby inhibiting a second signal, and thereby alleviating the abnormalcondition. Preferably, such antagonists are antibodies. Most preferably,such antibodies are chimeric and/or humanised to minimise theirimmunogenicity, as described previously.

In another approach, soluble forms of the polypeptide that retainbinding affinity for the ligand, substrate, enzyme, receptor, inquestion, may be administered. Typically, the polypeptide may beadministered in the form of fragments that retain the relevant portions.

In an alternative approach, expression of the gene encoding thepolypeptide can be inhibited using expression blocking techniques, suchas the use of antisense nucleic acid molecules (as described above),either internally generated or separately administered. Modifications ofgene expression can be obtained by designing complementary sequences orantisense molecules (DNA, RNA, or PNA) to the control, 5′ or regulatoryregions (signal sequence, promoters, enhancers and introns) of the geneencoding the polypeptide. Similarly, inhibition can be achieved using“triple helix” base-pairing methodology. Triple helix pairing is usefulbecause it causes inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature (Gee, J. E. et al. (1994) In: Huber, B.E. and B. I. Carr, Molecular and Immunologic Approaches, FuturaPublishing Co., Mt. Kisco, N.Y.). The complementary sequence orantisense molecule may also be designed to block translation of mRNA bypreventing the transcript from binding to ribosomes. Sucholigonucleotides may be administered or may be generated in situ fromexpression in vivo.

In addition, expression of the polypeptide of the invention may beprevented by using ribozymes specific to its encoding mRNA sequence.Ribozymes are catalytically active RNAs that can be natural or synthetic(see for example Usman, N, et al., Curr. Opin. Struct. Biol (1996) 6(4), 527-33). Synthetic ribozymes can be designed to specifically cleavemRNAs at selected positions thereby preventing translation of the mRNAsinto functional polypeptide. Ribozymes may be synthesised with a naturalribose phosphate backbone and natural bases, as normally found in RNAmolecules. Alternatively the ribozymes may be synthesised withnon-natural backbones, for example, 2′-O-methyl RNA, to provideprotection from ribonuclease degradation and may contain modified bases.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends of the moleculeor the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of non-traditional bases such asinosine, queosine and butosine, as well as acetyl-, methyl-, thio- andsimilarly modified forms of adenine, cytidine, guanine, thymine anduridine which are not as easily recognised by endogenous endonucleases.

For treating abnormal conditions related to an under-expression of thepolypeptide of the invention and its activity, several approaches arealso available. One approach comprises administering to a subject atherapeutically effective amount of a compound that activates thepolypeptide, i.e., an agonist as described above, to alleviate theabnormal condition. Alternatively, a therapeutic amount of thepolypeptide in combination with a suitable pharmaceutical carrier may beadministered to restore the relevant physiological balance ofpolypeptide.

Gene therapy may be employed to effect the endogenous production of thepolypeptide by the relevant cells in the subject. Gene therapy is usedto treat permanently the inappropriate production of the polypeptide byreplacing a defective gene with a corrected therapeutic gene.

Gene therapy of the present invention can occur in vivo or ex vivo. Exvivo gene therapy requires the isolation and purification of patientcells, the introduction of a therapeutic gene and introduction of thegenetically altered cells back into the patient. In contrast, in vivogene therapy does not require isolation and purification of a patient'scells.

The therapeutic gene is typically “packaged” for administration to apatient. Gene delivery vehicles may be non-viral, such as liposomes, orreplication-deficient viruses, such as adenovirus as described byBerkner, K. L., in Curr. Top. Microbiol. Immunol., 158, 39-66 (1992) oradeno-associated virus (AAV) vectors as described by Muzyczka, N., inCurr. Top. Microbiol. Immunol., 158, 97-129 (1992) and U.S. Pat. No.5,252,479. For example, a nucleic acid molecule encoding a polypeptideof the invention may be engineered for expression in areplication-defective retroviral vector. This expression construct maythen be isolated and introduced into a packaging cell transduced with aretroviral plasmid vector containing RNA encoding the polypeptide, suchthat the packaging cell now produces infectious viral particlescontaining the gene of interest. These producer cells may beadministered to a subject for engineering cells in vivo and expressionof the polypeptide in vivo (see Chapter 20, Gene Therapy and otherMolecular Genetic-based Therapeutic Approaches, (and references citedtherein) in Human Molecular Genetics (1996), T Strachan and A P Read,BIOS Scientific Publishers Ltd).

Another approach is the administration of “naked DNA” in which thetherapeutic gene is directly injected into the bloodstream or muscletissue.

In situations in which the polypeptides or nucleic acid molecules of theinvention are disease-causing agents, the invention provides that theycan be used in vaccines to raise antibodies against the disease causingagent. Where the aforementioned polypeptide or nucleic acid molecule isone that is up-regulated, vaccine development can involve the raising ofantibodies or T cells against such agents (as described in WO00/29428).

Vaccines according to the invention may either be prophylactic (ie. toprevent infection) or therapeutic (ie. to treat disease afterinfection). Such vaccines comprise immunising antigen(s), immunogen(s),polypeptide(s), protein(s) or nucleic acid, usually in combination withpharmaceutically-acceptable carriers as described above, which includeany carrier that does not itself induce the production of antibodiesharmful to the individual receiving the composition. Additionally, thesecarriers may function as immunostimulating agents (“adjuvants”).Furthermore, the antigen or immunogen may be conjugated to a bacterialtoxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori,and other pathogens.

Since polypeptides may be broken down in the stomach, vaccinescomprising polypeptides are preferably administered parenterally (forinstance, subcutaneous, intramuscular, intravenous, or intradermalinjection). Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the recipient, and aqueous andnon-aqueous sterile suspensions which may include suspending agents orthickening agents.

The vaccine formulations of the invention may be presented in unit-doseor multi-dose containers. For example, sealed ampoules and vials and maybe stored in a freeze-dried condition requiring only the addition of thesterile liquid carrier immediately prior to use. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

Genetic delivery of antibodies that bind to polypeptides according tothe invention may also be effected, for example, as described inInternational patent application WO98/55607.

The technology referred to as jet injection (see, for example,www.powderject.com) may also be useful in the formulation of vaccinecompositions.

A number of suitable methods for vaccination and vaccine deliverysystems are described in International patent application WO00/29428.

This invention also relates to the use of nucleic acid moleculesaccording to the present invention as diagnostic reagents. Detection ofa mutated form of the gene characterised by the nucleic acid moleculesof the invention which is associated with a dysfunction will provide adiagnostic tool that can add to, or define, a diagnosis of a disease, orsusceptibility to a disease, which results from under-expression,over-expression or altered spatial or temporal expression of the gene.Individuals carrying mutations in the gene may be detected at the DNAlevel by a variety of techniques.

Nucleic acid molecules for diagnosis may be obtained from a subject'scells, such as from blood, urine, saliva, tissue biopsy or autopsymaterial. The genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR, ligase chain reaction (LCR),strand displacement amplification (SDA), or other amplificationtechniques (see Saiki et al., Nature, 324, 163-166 (1986); Bej, et al.,Crit. Rev. Biochem. Molec. Biol., 26, 301-334 (1991); Birkenmeyer etal., J. Virol. Meth., 35, 117-126 (1991); Van Brunt, J., Bio/Technology,8, 291-294 (1990)) prior to analysis.

In one embodiment, this aspect of the invention provides a method ofdiagnosing a disease in a patient, comprising assessing the level ofexpression of a natural gene encoding a polypeptide according to theinvention and comparing said level of expression to a control level,wherein a level that is different to said control level is indicative ofdisease. The method may comprise the steps of:

-   a) contacting a sample of tissue from the patient with a nucleic    acid probe under stringent conditions that allow the formation of a    hybrid complex between a nucleic acid molecule of the invention and    the probe;-   b) contacting a control sample with said probe under the same    conditions used in step a);-   c) and detecting the presence of hybrid complexes in said samples;    wherein detection of levels of the hybrid complex in the patient    sample that differ from levels of the hybrid complex in the control    sample is indicative of disease.

A further aspect of the invention comprises a diagnostic methodcomprising the steps of:

-   a) obtaining a tissue sample from a patient being tested for    disease;-   b) isolating a nucleic acid molecule according to the invention from    said tissue sample; and-   c) diagnosing the patient for disease by detecting the presence of a    mutation in the nucleic acid molecule which is associated with    disease.

To aid the detection of nucleic acid molecules in the above-describedmethods, an amplification step, for example using PCR, may be included.

Deletions and insertions can be detected by a change in the size of theamplified product in comparison to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to labelled RNA of theinvention or alternatively, labelled antisense DNA sequences of theinvention. Perfectly-matched sequences can be distinguished frommismatched duplexes by RNase digestion or by assessing differences inmelting temperatures. The presence or absence of the mutation in thepatient may be detected by contacting DNA with a nucleic acid probe thathybridises to the DNA under stringent conditions to form a hybriddouble-stranded molecule, the hybrid double-stranded molecule having anunhybridised portion of the nucleic acid probe strand at any portioncorresponding to a mutation associated with disease; and detecting thepresence or absence of an unhybridised portion of the probe strand as anindication of the presence or absence of a disease-associated mutationin the corresponding portion of the DNA strand.

Such diagnostics are particularly useful for prenatal and even neonataltesting.

Point mutations and other sequence differences between the referencegene and “mutant” genes can be identified by other well-knowntechniques, such as direct DNA sequencing or single-strandconformational polymorphism, (see Orita et al., Genomics, 5, 874-879(1989)). For example, a sequencing primer may be used withdouble-stranded PCR product or a single-stranded template moleculegenerated by a modified PCR. The sequence determination is performed byconventional procedures with radiolabelled nucleotides or by automaticsequencing procedures with fluorescent-tags. Cloned DNA segments mayalso be used as probes to detect specific DNA segments. The sensitivityof this method is greatly enhanced when combined with PCR. Further,point mutations and other sequence variations, such as polymorphisms,can be detected as described above, for example, through the use ofallele-specific oligonucleotides for PCR amplification of sequences thatdiffer by single nucleotides.

DNA sequence differences may also be detected by alterations in theelectrophoretic mobility of DNA fragments in gels, with or withoutdenaturing agents, or by direct DNA sequencing (for example, Myers etal., Science (1985) 230:1242). Sequence changes at specific locationsmay also be revealed by nuclease protection assays, such as RNase and S1protection or the chemical cleavage method (see Cotton et al., Proc.Natl. Acad. Sci. USA (1985) 85:4397-4401).

In addition to conventional gel electrophoresis and DNA sequencing,mutations such as microdeletions, aneuploidies, translocations,inversions, can also be detected by in situ analysis (see, for example,Keller et al., DNA Probes, 2nd Ed., Stockton Press, New York, N.Y., USA(1993)), that is, DNA or RNA sequences in cells can be analysed formutations without need for their isolation and/or immobilisation onto amembrane. Fluorescence in situ hybridization (FISH) is presently themost commonly applied method and numerous reviews of FISH have appeared(see, for example, Trachuck et al., Science, 250, 559-562 (1990), andTrask et al., Trends, Genet., 7, 149-154 (1991)).

In another embodiment of the invention, an array of oligonucleotideprobes comprising a nucleic acid molecule according to the invention canbe constructed to conduct efficient screening of genetic variants,mutations and polymorphisms. Array technology methods are well known andhave general applicability and can be used to address a variety ofquestions in molecular genetics including gene expression, geneticlinkage, and genetic variability (see for example: M. Chee et al.,Science (1996), Vol 274, pp 610-613).

In one embodiment, the array is prepared and used according to themethods described in PCT application WO95/11995 (Chee et al); Lockhart,D. J. et al. (1996) Nat. Biotech. 14: 1675-1680); and Schena, M. et al.(1996) Proc. Natl. Acad. Sci. 93: 10614-10619). Oligonucleotide pairsmay range from two to over one million. The oligomers are synthesized atdesignated areas on a substrate using a light-directed chemical process.The substrate may be paper, nylon or other type of membrane, filter,chip, glass slide or any other suitable solid support. In anotheraspect, an oligonucleotide may be synthesized on the surface of thesubstrate by using a chemical coupling procedure and an ink jetapplication apparatus, as described in PCT application WO95/25116(Baldeschweiler et al.). In another aspect, a “gridded” array analogousto a dot (or slot) blot may be used to arrange and link cDNA fragmentsor oligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any other numberbetween two and over one million which lends itself to the efficient useof commercially-available instrumentation.

In addition to the methods discussed above, diseases may be diagnosed bymethods comprising determining, from a sample derived from a subject, anabnormally decreased or increased level of polypeptide or mRNA.Decreased or increased expression can be measured at the RNA level usingany of the methods well known in the art for the quantitation ofpolynucleotides, such as, for example, nucleic acid amplification, forinstance PCR, RT-PCR, RNase protection, Northern blotting and otherhybridization methods.

Assay techniques that can be used to determine levels of a polypeptideof the present invention in a sample derived from a host are well-knownto those of skill in the art and are discussed in some detail above(including radioimmunoassays, competitive-binding assays, Western Blotanalysis and ELISA assays). This aspect of the invention provides adiagnostic method which comprises the steps of: (a) contacting a ligandas described above with a biological sample under conditions suitablefor the formation of a ligand-polypeptide complex; and (b) detectingsaid complex.

Protocols such as ELISA, RIA, and FACS for measuring polypeptide levelsmay additionally provide a basis for diagnosing altered or abnormallevels of polypeptide expression. Normal or standard values forpolypeptide expression are established by combining body fluids or cellextracts taken from normal mammalian subjects, preferably humans, withantibody to the polypeptide under conditions suitable for complexformation The amount of standard complex formation may be quantified byvarious methods, such as by photometric means.

Antibodies which specifically bind to a polypeptide of the invention maybe used for the diagnosis of conditions or diseases characterised byexpression of the polypeptide, or in assays to monitor patients beingtreated with the polypeptides, nucleic acid molecules, ligands and othercompounds of the invention. Antibodies useful for diagnostic purposesmay be prepared in the same manner as those described above fortherapeutics. Diagnostic assays for the polypeptide include methods thatutilise the antibody and a label to detect the polypeptide in human bodyfluids or extracts of cells or tissues. The antibodies may be used withor without modification, and may be labelled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules known in the art may be used, several of which aredescribed above.

Quantities of polypeptide expressed in subject, control and diseasesamples from biopsied tissues are compared with the standard values.Deviation between standard and subject values establishes the parametersfor diagnosing disease. Diagnostic assays may be used to distinguishbetween absence, presence, and excess expression of polypeptide and tomonitor regulation of polypeptide levels during therapeuticintervention. Such assays may also be used to evaluate the efficacy of aparticular therapeutic treatment regimen in animal studies, in clinicaltrials or in monitoring the treatment of an individual patient.

A diagnostic kit of the present invention may comprise:

-   (a) a nucleic acid molecule of the present invention;-   (b) a polypeptide of the present invention; or-   (c) a ligand of the present invention.

In one aspect of the invention, a diagnostic kit may comprise a firstcontainer containing a nucleic acid probe that hybridises understringent conditions with a nucleic acid molecule according to theinvention; a second container containing primers useful for amplifyingthe nucleic acid molecule; and instructions for using the probe andprimers for facilitating the diagnosis of disease. The kit may furthercomprise a third container holding an agent for digesting unhybridisedRNA.

In an alternative aspect of the invention, a diagnostic kit may comprisean array of nucleic acid molecules, at least one of which may be anucleic acid molecule according to the invention.

To detect polypeptide according to the invention, a diagnostic kit maycomprise one or more antibodies that bind to a polypeptide according tothe invention; and a reagent useful for the detection of a bindingreaction between the antibody and the polypeptide.

Such kits will be of use in diagnosing a disease or susceptibility todiseases in which metalloproteases are implicated, particularlyrespiratory disorders, including emphysema and cystic fibrosis,metabolic disorders, cardiovascular disorders, bacterial infections,hypertension, proliferative disorders, including cancer,autoimmune/inflammatory disorders, including rheumatoid arthritis,neurological disorders, developmental disorders and reproductivedisorders.

Various aspects and embodiments of the present invention will now bedescribed in more detail by way of example, with particular reference toINSP005 polypeptides.

It will be appreciated that modification of detail may be made withoutdeparting from the scope of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Summary of results of database searches using the INSP005predicted polypeptide sequence as a query sequence (sequence alignmentsshown) (SEQ ID NO:52, identified as “Query” and SEQ ID NO:53, identifiedas “Sbjct” for Blastp vs. NCBI-nr; SEQ ID NO:54, identified as “Query”and SEQ ID NO:55, identified as “Sbjct” for Tblastn vs. NCBI-est).

FIG. 2: Table of human cDNA libraries used in the INSP005 cloninginvestigation.

FIG. 3: Nucleotide sequence of the INSP005 predicted polypeptide andpredicted amino acid sequence (SEQ ID NO:56).

FIG. 4: Table of INSP005 cloning primers.

FIG. 5: 3′nucleotide and amino acid sequence of INSP005 identified byRACE PCR (SEQ ID NO:57).

FIG. 6: Table of primers used during INSP005 sequencing.

FIG. 7: Putative full-length INSP005a cloned from human uterus cDNA (SEQID NO:58).

FIG. 8: INSP005a blastp vs. NCBI-nr database (top ten hits and toprelated alignment shown) (SEQ ID NO:59, identified as “Query” and SEQ IDNO:60, identified as “Sbjct”).

FIG. 9: Map of PCR4-TOPO-IPAAAIPAAA7883-1 INSP005a cloning plasmid.

FIG. 10: Putative full-length INSP005b (SEQ ID NO:61) cloned from a poolof cDNAs derived from human primary lung fibroblasts, keratinocytes andosteoarthritis synovium.

FIG. 11: INSP005b blastp vs. NCBI-nr database (top ten hits and toprelated alignment shown) (SEQ ID NO:62, identified as “Query” and SEQ IDNO:63, identified as “Sbjct”).

FIG. 12: Map of PCR-TOPO-IPAAA78836-2 INSP005b cloning plasmid.

FIG. 13: Multiple alignment of the INSP005 predicted polypeptidesequence (SEQ ID NO:66), the INSP005a cloned polypeptide sequence (SEQID NO:14), the INSP005b cloned polypeptide sequence (SEQ ID NO:34) andcertain prior art sequences of interest (SEQ ID NO:64, WO2002/16566-A2and SEQ ID NO:65, AX526191).

FIG. 14: SignalP signal peptide prediction data for the INSP005bpolypeptide.

FIG. 15A: Effect of hIL-6 or INSP005b plasmid delivery on serum ASATlevels.

FIG. 15B: Effect of hIL-6 or INSP005b plasmid delivery on serum ALATlevels.

FIG. 16A: Effect of hIL-6 or INSP005b plasmid delivery on serum mIL-6levels.

FIG. 16B: Effect of hIL-6 or INSP005b plasmid delivery on serumTNF-alpha levels.

EXAMPLES Example 1 INSP005 Predicted Polypeptide

An INSP005 polypeptide sequence (SEQ ID NO:37) predicted by proprietarybioinformatics techniques was used as a query sequence for searches ofthe following databases:

NCBI-nr NCBI-nt NCBI-pat-aa NCBI-pat-nt NCBI-month-aa NCBI-month-ntNCBI-est

The results of these searches are summarised in FIG. 1, which shows tworelevant sequence alignments. The headings in FIG. 1 indicate whichsearching/alignment algorithms were used and which database wassearched. These results show that the closest related match to theINSP005 predicted polypeptide sequence is the hatching enzyme EHE4 fromAnguilla japonica (Japanese eel). These searches also identified threeother prior art sequences of interest, which are discussed in moredetail below.

Members of the choriolysin/astacin-like family of metalloproteases havebeen implicated in chorion hardening of oviparous fish eggs afterfertilisation (for an example see Shibata et al. (2000) J. Biol. Chemvol. 275, No. 12 p 8349). This post-fertilisation change preventspolyspermy and corresponds to the formation of fertilisation membranesin sea urchin, amphibian and the zona reaction in mammals. They havealso been implicated in the hydrolysis of the hardened chorion at thetime of hatching and the hydrolysis of unfertilised egg chorions.

As described above, the identification of novel metalloproteases is ofextreme importance in increasing understanding of the underlyingpathways that lead to certain disease states in which these proteins areimplicated, and in developing more effective gene or drug therapies totreat these disorders. Similarly, the identification of further membersof the astacin/choriolysin-like family of metalloproteases is of extremeimportance in increasing understanding of the underlying pathways thatlead to certain disease states in which these proteins are implicated,and in developing more effective gene or drug therapies to treat thesedisorders.

Example 2 Summary of INSP005 Cloning

1.1 cDNA Libraries

Human cDNA libraries (in bacteriophage lambda (λ) vectors) werepurchased from Stratagene or Clontech or prepared at the SeronoPharmaceutical Research Institute in λ ZAP or λ GT10 vectors accordingto the manufacturer's protocol (Stratagene). Bacteriophage λ DNA wasprepared from small-scale cultures of infected E. coli host strain usingthe Wizard Lambda Preps DNA purification system according to themanufacturer's instructions (Promega, Corporation, Madison Wis.). Thelist of libraries and host strains used is shown in FIG. 2. Eight pools(A-H) of five different libraries (100 ng/μl phage DNA) were used insubsequent PCR reactions.

1.2 Generation of Reverse Transcribed cDNA Templates

Total RNA was isolated from primary human cells, human cell lines andhuman tissues using the Trizol™ reagent (Invitrogen) according to themanufacturer's instructions or purchased from Clontech, Invitrogen orAmbion. The quality and concentration of the RNA was analysed using anAgilent 2100 Bioanalyzer.

For cDNA synthesis the reaction mixture contained: 1 μl oligo (dT)₁₅primer (500 μg/ml, Promega cat. no. C 1101), 2 μg total RNA, 1 μl 10 mMdNTPs in a volume of 12 μl. The mixture was heated to 65° C. for 5 minand then chilled on ice. The following reagents were then added: 4 μl 5×first strand buffer, 2 μl DTT (0.1M), 1 μl RNAseOut recombinantribonuclease inhibitor (40 units/μl, Promega, cat. no. N 2511) andincubated at 42° C. for 2 min before addition of 1 μl (200 units) ofSuperscript II (Invitrogen cat. no. 18064-014). The mixture wasincubated at 42° C. for 50 min and then heated at 70° C. for 15 min. Toremove the RNA template, 1 μl (2 units) of E. coli RNase H (Invitrogencat. no. 18021-014) was added and the reaction mixture further incubatedat 37° C. for 20 min. The final reaction mix was diluted to 200 μl withsterile water and stored at −80° C. cDNA pools were generated by mixingequal volumes of 5 different cDNA templates.

1.3 PCR of Virtual cDNAs from Phage Library DNA

A partial cDNA encoding INSP005 was obtained as a PCR amplificationproduct of 248 bp using gene specific cloning primers (CP1 and CP2, FIG.3 and FIG. 4). The PCR was performed in a final volume of 50 μlcontaining 1× AmpliTaq™ buffer, 200 μM dNTPs, 50 pmoles each of cloningprimers, 2.5 units of AmpliTaq™ (Perkin Elmer) and 100 ng of each phagelibrary pool DNA using an MJ Research DNA Engine, programmed as follows:94° C., 1 min; 40 cycles of 94° C., 1 min, x° C., and y min and 72° C.,(where x is the lowest Tm—5° C. and y=1 min per kb of product); followedby 1 cycle at 72° C. for 7 min and a holding cycle at 4° C.

The amplification products were visualized on 0.8 % agarose gels in 1×TAE buffer (Invitrogen) and PCR products migrating at the predictedmolecular mass were purified from the gel using the Wizard PCR Preps DNAPurification System (Promega). PCR products eluted in 50 μl of sterilewater were either subcloned directly or stored at −20° C.

1.4 Gene Specific Cloning Primers for PCR

Pairs of PCR primers having a length of between 18 and 25 bases weredesigned for amplifying the full length and partial sequence of thevirtual cDNA using Primer Designer Software (Scientific & EducationalSoftware, PO Box 72045, Durham, N.C. 27722-2045, USA). PCR primers wereoptimized to have a Tm close to 55±10° C. and a GC content of 40-60%.Primers were selected which had high selectivity for the target sequenceINSP005 (little or no non-specific priming).

1.5 Subcloning of PCR Products

PCR products were subcloned into the topoisomerase I modified cloningvector (PCRII TOPO) using the TA cloning kit purchased from theInvitrogen Corporation using the conditions specified by themanufacturer. Briefly, 4 μl of gel purified PCR product from the humanlibrary pool N amplification was incubated for 15 min at roomtemperature with 1 μl of TOPO vector and 1 μl salt solution. Thereaction mixture was then transformed into E. coli strain TOP10(Invitrogen) as follows: a 50 μl aliquot of One Shot TOP10 cells wasthawed on ice and 2 μl of TOPO reaction was added. The mixture wasincubated for 15 min on ice and then heat shocked by incubation at 42°C. for exactly 30 s. Samples were returned to ice and 250 μl of warm SOCmedia (room temperature) was added. Samples were incubated with shaking(220 rpm) for 1 h at 37° C. The transformation mixture was then platedon L-broth (LB) plates containing ampicillin (100 μg/ml) and incubatedovernight at 37° C. Ampicillin resistant colonies containing cDNAinserts were identified by colony PCR.

1.6 Colony PCR

Colonies were inoculated into 50 μl sterile water using a steriletoothpick. A 10 μl aliquot of the inoculum was then subjected to PCR ina total reaction volume of 20 μl as described above, except the primersused were SP6 and T7. The cycling conditions were as follows: 94° C., 2min; 30 cycles of 94° C., 30 sec, 47° C., 30 sec and 72° C. for 1 cycle,72° C., 7 min. Samples were then maintained at 4° C. (holding cycle)before further analysis.

PCR reaction products were analyzed on 1% agarose gels in 1× TAE buffer.Colonies which gave the expected PCR product size (248 bp cDNA+185 bpdue to the multiple cloning site or MCS) were grown up overnight at 37°C. in 5 ml L-Broth (LB) containing ampicillin (100 μg/ml), with shakingat 220 rpm at 37° C.

1.7 Plasmid DNA Preparation and Sequencing

Miniprep plasmid DNA was prepared from 5 ml cultures using a QiaprepTurbo 9600 robotic system (Qiagen) or Wizard Plus SV Minipreps kit(Promega cat. no. 1460) according to the manufacturer's instructions.Plasmid DNA was eluted in 100 μl of sterile water. The DNA concentrationwas measured using an Eppendorf BO photometer. Plasmid DNA (200-500 ng)was subjected to DNA sequencing with T7 primer and SP6 primer using theBigDyeTerminator system (Applied Biosystems cat. no. 4390246) accordingto the manufacturer's instructions. Sequencing reactions were purifiedusing Dye-Ex columns (Qiagen) or Montage SEQ 96 cleanup plates(Millipore cat. no. LSKS09624) then analyzed on an Applied Biosystems3700 sequencer.

1.8 Identification of the Full Length Sequence of INSP005 Using RACEPCR.

The predicted sequence of the INSP005 ORF is shown in FIG. 3. Attemptsto isolate the full length coding sequence by PCR failed on thelibraries tested, using primer pairs to amplify the full lengthprediction or a shorter version which uses a 2^(nd) predicted start siteat M96 in the open reading frame. The closest related sequences toINSP005 are the astacin-like metallopeptidase in Anguilla japonica andchoriolysin H in Oryzias latipes. INSP005 appears to be a humanorthologue of choriolysin H. Choriolysins are implicated in chorionhardening of oviparous fish eggs after fertilization, suggesting thatuterus may be a suitable source of the INSP005 mRNA. The choice of thistissue was further supported by the finding of a single EST, B1061462derived from a human uterus tumour.

In order to identify the full coding sequence, RACE PCR was performed oncDNA prepared from uterus RNA (purchased from Clontech) using theGeneRacer kit (Invitrogen cat no. L1502-01) according to themanufacturer's instructions. For amplification of 3′ ends, the first PCRwas performed in a 50 μl reaction volume containing 1 μl RACE ReadycDNA, 5 μl of 10× High Fidelity buffer, 1 μl of dNTPs (10 mM), 2 μl of50 mM MgSO₄, 3 μl of GeneRacer 3′ primer (10 μM), 1 μl of gene specificprimer (78836-GR1-3′) (10 μM) and 2.5 units (0.5 μl) of Platinum Taq DNApolymerase Hi Fi (Invitrogen). The cycling conditions were as follows:94° C., 2 min; 5 cycles of 94° C. 30 s and 72° C. 2 min; 5 cycles of 94°C., 30 s and 70° C., 5 min; 25 cycles of 94° C., 30 s, 65° C. 30 s and68° C. 5 min; a final extension at 68° C. for 10 min and a holding cycleof 4° C. One μl of the amplification reaction was then used as atemplate for a nested PCR which was performed in a final reaction volumeof 50 μl with the same reagents as above except for the primers. Theprimers for the nested PCR were 1 μl of GeneRacer 3′ nested primer (10μM) and 1 μl of nested gene specific primer (78836-GR1nest-3′) (10 μM).The cycling conditions were 94° C., 2 min; 25 cycles of 94° C., 30 s,65° C., 30 s and 68° C., 5 min; a final extension at 68° C. for 10 minand a holding cycle of 4° C. PCR products were gel purified, subclonedinto pCR4-TOPO vector and sequenced as described above. All primers usedare listed in FIG. 4. The nucleotide sequence and amino acid sequence ofthe 3′ RACE product is shown in FIG. 5. The amino acid sequence encodedby the 3′ RACE product has an extended C-terminal, diverging from theprediction after nucleotide position 85 which was suggestive of analternatively spliced form.

1.9 Cloning of the Full Length Coding Sequence of INSP005 by PCR

The putative full length coding sequence of INSP005 was cloned fromhuman uterus cDNA (prepared as described in section 1.2) by PCR in a 50μl PCR reaction mixture as containing 2 μl uterus cDNA, 5 μl of 10× HighFidelity buffer, 1 μl of dNTPs (10 mM), 2 μl of 50 mM MgSO₄, 1 μl ofgene specific primer 78836-FL-F (10 μM), 1 μl of reverse gene specificprimer 78836-FL-R (10 μM) and 2.5 units (0.5 μl) of Platinum Taq DNApolymerase Hi Fi (Invitrogen). The cycling conditions were 94° C., 2min; 40 cycles of 94° C., 30 s, 55° C., 30 s and 68° C., 1 min 30 s min;a final extension at 68° C. for 10 min and a holding cycle of 4° C. Theamplification products were visualized on 0.8 % agarose gels in 1× TAEbuffer (Invitrogen) and PCR products migrating at the predictedmolecular mass (1048 bp) were purified from the gel using the Wizard PCRPreps DNA Purification System (Promega). PCR products were eluted in 50μl of sterile water and subcloned into pCR4 TOPO vector as described insection 1.4. Several ampicillin resistant colonies were subjected tocolony PCR as described in section 1.5 except that the extension time inthe amplification reaction was 2 min. Colonies containing the correctsize insert (1048 bp+99 bp due to the MCS) were grown up overnight at37° C. in 5 ml L-Broth (LB) containing ampicillin (100 μg/ml), withshaking at 220 rpm at 37° C. Miniprep plasmid DNA was prepared from 5 mlcultures using a Qiaprep Turbo 9600 robotic system (Qiagen) or WizardPlus SV Minipreps kit (Promega cat. no. 1460) according to themanufacturer's instructions and 200-500 ng of mini-prep DNA wassequenced as described in section 1.7 with T3 and T7 primers (FIG. 6).The cloned sequence is given in FIG. 7. The amino acid alignment of thecloned sequence (INSP005a) with the predicted sequence is shown in FIG.13. The map of the resultant plasmid, pCR4-TOPO-IPAAA78836-1 (SEQ IDNO:38; plasmid ID. No. 13164) is shown in FIG. 9.

2.0 Identification of cDNA Libraries/Templates Containing INSP005

PCR products obtained with CP1 and CP2 and migrating at the correct size(248 bp) were identified in library pool N (libraries 18, 19, 20 and21). A cDNA encoding a putative full length INSP005 (INSP005a) wasisolated from uterus cDNA using 78836-FL-F and 78836-FL-R primers.Primer 78836-FL-F is located in exon 3 of the predicted sequence. No PCRproducts were obtained using the reverse primer (78836-FL-R) withprimers located in exon 1 of the prediction.

A second putative full length version of INSP005 (INSP005b) containingan alternative 5′ end was cloned from a pool of cDNAs derived from humanprimary lung fibroblasts, keratinocytes and osteoarthritis synoviumusing primers 78836-FL2-F and 78836-FL-R but was not detected in uterus.The resultant PCR product (1313 bp—FIG. 10) was subcloned into pCR4 TOPOvector using the TOPO-TA cloning kit and sequenced as described insections 1.5-1.7. The map of the resultant plasmid,pCR4-TOPO-IPAAA78836-2 (SEQ ID NO:39; plasmid ID. No. 13296) is shown inFIG. 12.

2.1 Summary of Cloning Results

Attempts to clone the full-length INSP005 predicted polypeptideidentified two variants of the INSP005 predicted polypeptide, hereinreferred to as INSP005a and INSP005b (FIG. 13; SEQ ID NO:14 and SEQ IDNO:34, respectively). As described above, the INSP005a and INSP005bpolypeptides (and the INSP005b mature polypeptide) are herein referredto as the INSP005 polypeptides, as distinct from the INSP005 predictedpolypeptide.

The nucleotide and amino acid sequences for the predicted exons withinthe INSP005a and INSP005b polypeptides are given in SEQ ID NOs 1-12 andSEQ ID NOs 15-32, respectively. As described above, the putativefull-length nucleotide sequences of the INSP005a and INSP005bpolypeptides are given in SEQ ID NOs 13 and 33, respectively. The aminoacid sequences of the INSP005a and INSP005b polypeptides are given inSEQ ID NOs 14 and 34, respectively.

The relationships between the INSP005a and INSP005b polypeptides and theINSP005 predicted polypeptide and three prior art sequences of interestare shown in FIG. 13, which provides a sequence-level alignment of thesequences. These relationships will now be described in detail.

INSP005a is a putative full-length version of the INSP005 predictedpolypeptide from a uterus cDNA library. This sequence differs from theoriginal INSP005 prediction in that it has a truncated 5′ end, startingat methionine 3 of the original INSP005 predicted polypeptide (see FIG.13). INSP005a also has an extended 3′ end that incorporates an extraexon relative to the INSP005 predicted polypeptide. INSP005a has sixpredicted exons in total. These differences were not predicted due tothe low homology of those sequence elements to other metalloproteinases.In addition, there is an alternative amino acid used at position 22 ofINSP005a compared to the INSP005 predicted polypeptide. INSP005a is notpredicted to contain a signal peptide. INSP005a has no in framealternative upstream start methionine before an upstream STOP codon.

The polypeptide sequence shown in SEQ ID NO:14 (INSP005a), was used as aBLAST query against the NCBI non-redundant sequence database. The topten hits are all egg hatching-related enzymes from Anguilla japonica orchoriolytic proteases and align to the query sequence with highlysignificant E-values (from e⁻¹¹⁵ to 2e⁻⁴¹) (FIG. 8). FIG. 8 also showsthe alignment of the INSP005a polypeptide query sequence to the sequenceof the top biochemically annotated hit, the hatching enzyme HE13 fromAnguilla japonica. These results provide strong evidence that theINSP005a polypeptide is a metalloprotease, more specifically that it isa choriolysin/astacin-like metalloprotease.

INSP005b is a putative full-length version of the INSP005 predictedpolypeptide cloned from a pool of cDNAs derived from human primary lungfibroblasts, keratinocytes and osteoarthritis synovium. INSP005bsubsumes the original INSP005 predicted polypeptide sequence, though twoalternative amino acids are used at positions 117 and 222. It alsocontains three new upstream exons and one downstream exon, makingINSP005b a nine exon polypeptide. The final exon is shared withINSP005a. INSP005b was not detected in uterus. These differences werenot predicted due to the low homology of those sequence elements toother metalloproteinases. As described above, INSP005b is predicted tocontain a signal peptide with a cleavage site between amino acids 23 and24 (SEQ ID NOs 35 and 36; FIG. 14).

The polypeptide sequence shown in SEQ ID NO:34 (INSP005b), was used as aBLAST query against the NCBI non-redundant sequence database. The topten hits are all egg hatching-related enzymes from Anguilla japonica orchoriolytic proteases and align to the query sequence with highlysignificant E-values (from e⁻¹⁵² to 4e⁻⁴⁶) (FIG. 11). FIG. 11 also showsthe alignment of the cloned polypeptide query sequence to the sequenceof the top biochemically annotated hit, the hatching enzyme HE13 fromAnguilla japonica. These results provide strong evidence that theINSP005b polypeptide is a metalloprotease, more specifically that it isa choriolysin/astacin-like metalloprotease.

The first 7 exons of INSP005b match a nucleotide sequence disclosed inWO200216566-A2, given accession number AX443328 (see FIG. 1 and FIG.13), although the final 3′ exon is not disclosed in WO200216566-A2(Applera Corp). The nucleotide and polypeptide molecules of the presentinvention specifically exclude those disclosed in WO200216566-A2.

A further prior art sequence of interest is a spliced EST (BI061462.1;see FIG. 1) from uterus tumour covering exon 1 of INSP005a and exons 2,3 and 4 of INSP005b. The direction of the EST is not given in the reportand it is hard to come to a conclusion about the presence of a startmethionine from the translation. However, the nucleotide and polypeptidemolecules of the present invention specifically exclude the sequencesdisclosed in EST BI061462.1.

Another prior art sequence of interest, with accession number AX526191(Lexicon) (FIGS. 1 and 13), is described as cDNA in the relevantdatabase entry (disclosed in WO02/066624) and no reference is made to apossible reproductive role. It subsumes INSP005a and exons 2-8 ofINSP005b. However, an alternative amino acid is used at position 127 inINSP005a compared to the corresponding amino acid in INSP005b and theAX526191 (Lexicon) sequence. The start methionine of AX526191 is coveredby the uterus tumour EST described above. A signal peptide is predictedfor AX526191with a probability of 0.875. The nucleotide and polypeptidemolecules of the present invention do not include the sequencesdisclosed in WO02/066624, including that with accession number AX526191.

FIG. 13 also highlights the active site residues, which are identical ineach of the polypeptides shown. This provides further compellingevidence that the INSP005a and INSP005b polypeptides aremetalloproteases.

The INSP005a and INSP005b polypeptides therefore represent novelmetalloproteases, and there is strong evidence that they are members ofthe choriolysin/astacin-like family of metalloproteases. The INSP005aand INSP005b polypeptides may therefore play important roles inphysiological and pathological processes in humans, particularly inreproductive processes.

Example 3 Expression and Purification of the Cloned, His-tagged INSP005b

3.1 Expression

Human Embryonic Kidney 293 cells expressing the Epstein-Barr virusNuclear Antigen (HEK293-EBNA, Invitrogen) were maintained in suspensionin Ex-cell VPRO serum-free medium (seed stock, maintenance medium, JRH).Sixteen to 20 hours prior to transfection (Day-1), cells were seeded in2× T225 flasks (50 ml per flask in DMEM/F12 (1:1) containing 2% FBSseeding medium (JRH) at a density of 2×10⁵ cells/ml). The next day(transfection: day 0) the transfection took place by using the JetPEI™reagent (2 μl/μg of plasmid DNA, PolyPlus-transfection). For each flask,113 μg of cDNA (plasmid No. 13403) was co-transfected with 2.3 μg of GFP(fluorescent reporter gene). The transfection mix was then added to the2× T225 flasks and incubated at 37° C. (5% CO₂) for 6 days. In order toincrease the amount of material, this procedure was repeated with twoextra flasks to generate 200 ml total. Confirmation of positivetransfection was carried out by qualitative fluorescence examination atday 6 (Axiovert 10 Zeiss).

On day 6 (harvest day), supernatants (200 ml) from the four flasks werepooled and centrifuged (4° C., 400 g) and placed into a pot bearing aunique identifier.

One aliquot (500 ul) was kept for QC of the 6His-tagged protein(internal bioprocessing QC). The corresponding delivery sheet can befound in T. Battle's notebook 11140 p 28.

For extra production purposes, batch 2 was produced in 500 ml spinnertransfection, as follows:

Human Embryonic Kidney 293 cells expressing the Epstein-Barr virusNuclear Antigen (HEK293-EBNA, Invitrogen) were maintained in suspensionin Ex-cell VPRO serum-free medium (seed stock, maintenance medium, JRH).On the day of transfection, cells were counted, centrifuged (low speed)and the pellet re-suspended into the desired volume of FEME medium (seebelow) supplemented with 1% FCS to yield a cell concentration of 1×E 6viable cells/ml. The #13403 cDNA was diluted at 2 mg/liter volume(co-transfected with 2% eGFP) in FEME (200 ml/liter volume).PolyEthyleneImine transfection agent (4 mg/liter volume) was then addedto the cDNA solution, vortexed and incubated at room temperature for 10minutes (generating the transfection Mix).

This transfection mix was then added to the spinner and incubated for 90minutes in a CO₂ incubator (5% CO₂ and 37° C.). Fresh FEME medium (1%FCS) was added after 90 minutes to double the initial spinner volume.The spinner was then incubated for 6 days. On day 6 (harvest day),spinner supernatant (500 ml) was centrifuged (4° C., 400 g) and placedinto a pot bearing a unique identifier with plasmid number andfermentation number.

One aliquot (500 μl) was kept for QC of the 6His-tagged protein(internal bioprocessing QC).

3.2 Purification Process

The 200 ml culture medium sample containing the recombinant protein witha C-terminal 6His tag was diluted to a final volume of 400 ml with coldbuffer A (50 mM NaH₂PO₄; 600 mM NaCl; 8.7% (w/v) glycerol, pH 7.5). Thesample was filtered through a 0.22 um sterile filter (Millipore, 500 mlfilter unit) and kept at 4° C. in a 500 ml sterile square media bottle(Nalgene).

The 500 ml culture medium sample was diluted to a final volume of 1000ml with cold buffer A. The sample was filtered through a 0.22 um sterilefilter (Millipore, 500 ml filter unit) and kept at 4° C. in a 1000 mlsterile square media bottle (Nalgene).

The purifications were performed at 4° C. on the VISION workstation(Applied Biosystems) connected to an automatic sample loader(Labomatic). The purification procedure was composed of two sequentialsteps, metal affinity chromatography on a Poros 20 MC (AppliedBiosystems) column charged with Ni ions (4.6×50 mm, 0.83 ml), followedby a buffer exchange on a Sephadex G-25 medium (Amersham Pharmacia) gelfiltration column (1.0×15 cm).

For the first chromatography step the metal affinity column wasregenerated with 30 column volumes of EDTA solution (100 mM EDTA; 1 MNaCl; pH 8.0), recharged with Ni ions through washing with 15 columnvolumes of a 100 mM NiSO₄ solution, washed with 10 column volumes ofbuffer A, followed by 7 column volumes of buffer B (50 mM NaH₂PO₄; 600mM NaCl; 8.7% (w/v) glycerol, 400 mM; imidazole, pH 7.5), and finallyequilibrated with 15 column volumes of buffer A containing 15 mMimidazole. The sample was transferred, by the Labomatic sample loader,into a 200 ml sample loop and subsequently charged onto the Ni metalaffinity column at a flow rate of 10 ml/min. The charging procedure wasrepeated 2 and 5 times, respectively in order to transfer the entiresample volume (400 or 1000 ml) onto the Ni column. The column wassubsequently washed with 12 column volumes of buffer A, followed by 28column volumes of buffer A containing 20 mM imidazole. During the 20 mMimidazole wash loosely attached contaminating proteins were elution ofthe column. The recombinant His-tagged protein was finally eluted with10 column volumes of buffer B at a flow rate of 2 ml/min, and the elutedprotein was collected in a 1.6 ml fraction.

For the second chromatography step, the Sephadex G-25 gel-filtrationcolumn was regenerated with 2 ml of buffer D (1.137 M NaCl; 2.7 mM KCl;1.5 mM KH₂PO₄; 8 mM Na₂HPO₄; pH 7.2), and subsequently equilibrated with4 column volumes of buffer C (137 mM NaCl; 2.7 mM KCl; 1.5 mM KH₂PO₄; 8mM Na₂HPO₄; 20% (w/v) glycerol; pH 7.4). The peak fraction eluted fromthe Ni-column was automatically through the integrated sample loader onthe VISION loaded onto the Sephadex G-25 column and the protein waseluted with buffer C at a flow rate of 2 ml/min. The protein sample fromthe Sephadex G-25 column was recovered in a 2.2 ml fraction. Thefraction was filtered through a 0.22 um sterile centrifugation filter(Millipore), frozen and stored at −80C. An aliquot of the sample wasanalyzed on SDS-PAGE (4-12% NuPAGE gel; Novex) by coomassie staining andWestern blot with anti-His antibodies.

Coomassie staining. The NuPAGE gel was stained in a 0.1% coomassie blueR250 staining solution (30% methanol, 10% acetic acid) at roomtemperature for 1 h and subsequently destained in 20% methanol, 7.5%acetic acid until the background was clear and the protein bands clearlyvisible.

Western blot. Following the electrophoresis the proteins wereelectrotransferred from the gel to a nitrocellulose membrane at 290 mAfor 1 hour at 4° C. The membrane was blocked with 5% milk powder inbuffer E (137 mM NaCl; 2.7 mM KCl; 1.5 mM KH₂PO₄; 8 mM Na₂HPO₄; 0.1%Tween 20, pH 7.4) for 1 h at room temperature, and subsequentlyincubated with a mixture of 2 rabbit polyclonal anti-His antibodies(G-18 and H-15, 0.2 ug/ml each; Santa Cruz) in 2.5% milk powder inbuffer E overnight at 4° C. After further 1 hour incubation at roomtemperature, the membrane was washed with buffer E (3×10 min), and thenincubated with a secondary HRP-conjugated anti-rabbit antibody (DAKO,HRP 0399) diluted 1/3000 in buffer E containing 2.5% milk powder for 2hours at room temperature. After washing with buffer E (3×10 minutes),the membrane was developed with the ECL kit (Amersham Pharmacia) for 1min. The membrane was subsequently exposed to a Hyperfilm (AmershamPharmacia), the film developed and the western blot image visuallyanalyzed.

Protein assay. The protein concentration was determined using the BCAprotein assay kit (Pierce) with bovine serum albumin as standard. 78 and90 μg purified protein was recovered from the 200 ml and 500 ml culturemedium samples, respectively.

Example 4 INSP005b in Mouse Model of Fulminant Liver Hepatitis

4.1 Introduction

In order to characterise INSP005b in vivo, the muscle electrotransfertechnique was used to express INSP005b protein in the circulation of WTand ConA treated animals. No significant changes in serum transaminaselevels or TNF-alpha, IFN-gamma, IL-6, IL-4 or MCP-1 cytokine levels wereobserved after electrotransfer of INSP005b in WT animals. Electroporatedanimals were then challenged with ConA in order to determine INSP005beffects on serum cytokine levels and transaminase levels.

4.2 Background—Concanavalin A (ConA)-induced Liver Hepatitis

Toxic liver disease represents a worldwide health problem in humans forwhich pharmacological treatments have yet to be discovered. For instanceactive chronic hepatitis leading to liver cirrhosis is a disease state,in which liver parenchymal cells are progressively destroyed byactivated T cells. ConA-induced liver toxicity is one of threeexperimental models of T-cell dependent apoptotic and necrotic liverinjury described in mice. Gal N (D-Galactosamine) sensitized micechallenged with either activating anti-CD3 monoclonal AB or withsuperantigen SEB develop severe apoptotic and secondary necrotic liverinjury (Kusters S, Gastroenterology. 1996 August; 111(2):462-71).Injection of the T-cell mitogenic plant lectin ConA to non-sensitizedmice results also in hepatic apoptosis that precedes necrosis. ConAinduces the release of systemic TNF-alpha and IFN-gamma and variousother cytokines. Both TNF-alpha and IFN-gamma are critical mediators ofliver injury. Transaminase release 8 hours after the insult indicatessevere liver destruction. Several cell types have been shown to beinvolved in liver damage, CD4 T cells, macrophages and natural killercells (Kaneko J Exp Med 2000, 191, 105-114). Anti-CD4 antibodies blockactivation of T cells and consequently liver damage (Tiegs et al. 1992,J Clin Invest 90, 196-203). Pre-treatment of mice with monoclonalantibodies against CD8 failed to protect, whereas deletion ofmacrophages prevented the induction of hepatitis.

The present study was undertaken to investigate the role of INSP005b, achoriolysin like protein, in ConA-induced liver hepatitis. Severalcytokines have been shown either to be critical in inducing or inconferring protection from ConA-induced liver damage. TNF-alpha forexample is one of the first cytokines produced after ConA injection andanti-TNF-alpha antibodies confer protection against disease (Seino etal. 2001, Annals of surgery 234, 681). IFN-gamma appears also to be acritical mediator of liver injury, since anti-IFN-gamma antiserumsignificantly protect mice, as measured by decreased levels oftransaminases in the blood of ConA-treated animals (see Kusters et al.,above). In liver injury, increased production of IFN-gamma was observedin patients with autoimmune or viral hepatitis. In addition transgenicmice expressing IFN-gamma in the liver develop liver injury resemblingchronic active hepatitis (Toyonaga et al. 1994, PNAS 91, 614-618).IFN-gamma may also be cytotoxic to hepatocytes, since in vitro IFN-gammainduces cell death in mouse hepatocytes that was accelerated by TNF(Morita et al. 1995, Hepatology 21, 1585-1593).

Other molecules have been described to be protective in the ConA model.A single administration of rhIL-6 completely inhibited the release oftransaminases (Mizuhara et al. 1994, J. Exp. Med. 179, 1529-1537).

4.3 cDNA Electrotransfer into Muscle Fibers in Order to Achieve SystemicExpression of a Protein of Interest

Among the non-viral techniques for gene transfer in vivo, the directinjection of plasmid DNA into the muscle and subsequent electroporationis simple, inexpensive and safe. The post-mitotic nature and longevityof myofibers permits stable expression of transfected genes, althoughthe transfected DNA does not usually undergo chromosomal integration(Somiari et al. 2000, Molecular Therapy 2, 178). Several reports havedemonstrated that secretion of muscle-produced proteins into the bloodstream can be achieved after electroporation of corresponding cDNAs(Rizzuto et al. PNAS, 1996, 6417; Aihara H et al., 1998, Nature Biotech16, 867). In addition in vivo efficacy of muscle expressed Epo andIL-18BP in disease models has been shown (Rizzuto, 2000, Human GeneTherapy 41, 1891; Mallat, 2001, Circulation research 89, 41).

4.4 Materials and Methods

4.4.1 Animals

In all of the studies male C57/BL6 male (8 weeks of age) were used. Ingeneral, 10 animals per experimental group are used. Mice weremaintained in standard conditions under a 12-hour light-dark cycle,provided irradiated food and water ad libitum.

4.4.2 Muscle Electrotransfer

4.4.2.1 Choice of Vector

His or StrepII tagged IL6 and INSP005b (IPAAA78836-2) genes were clonedin the Gateway compatible pDEST12.2 vector containing the CMV promoter.

4.4.2.2 Electroporation Protocol

Mice were anaesthetised with gas (isofluran Baxter, Ref: ZDG9623).Hindlimbs were shaved and an echo graphic gel was applied. Hyaluronidasewas injected in the posterior tibialis muscle with (20 U in 50 μlsterile NaCl 0.9% , Sigma Ref. H3631). After 10 min, 100 μg of plasmid(50 μg per leg in 25 μl of sterile NaCl 0.9%) was injected in the samemuscle. The DNA was prepared in the Buffer PBS-L-Glutamate (6 mg/ml;L-Glutamate Sigma P4761) before intramuscular injection. Forelectrotransfer, the electric field was applied for each leg with theElectroSquarePorator BTX ref ECM830 at 75 Volts during 20 ms for eachpulse, 10 pulses with an interval of 1 second in a unipolar way with 2round electrodes (size 0.5 mm diameter).

4.4.3 The ConA Model

4.4.3.1 ConA I.V. Injection and Blood Sampling

8 weeks old Female Mice C57/B16 were purchased from IFFA CREDO. ConA(Sigma ref. C7275) was injected at 18 mg/kg i.v. and blood samples weretaken at 1.30 and 8 hours post injection. At the time of sacrifice,blood was taken from the heart.

4.4.3.2 Detection of Cytokines and Transaminases in Blood Samples

IL2, IL5, IL4, TNF-alpha and IFN-gamma cytokine levels were measuredusing the TH1/TH2 CBA assay. TNF-alpha, IL-6, MCP1, IFN-alpha, IL-10 andIL-12 were detected using the Inflammation CBA assay. Transaminase bloodparameters were determined using the COBAS instrument (Hitachi).

4.4.3.3 INSP0005b and IL-6 Electrotransfer

At day 0 electrotransfer of pDEST12.2.INSP005b, pDEST12.2-hIL-6 as wellas and the empty vector control (electrotransfer protocol see above) wasperformed. At day 5 after electrotransfer, ConA (18 mg/kg) was injectedi.v. and blood sampled at 2 time points (1.30, 8 hours). Cytokine andASAT ALAT measurements were performed like described above.

4.5 Results

We have found that INSP005b protects from liver injury in a mouse modelmimicking fulminant hepatitis after systemic delivery of the proteinusing electrotransfer. FIGS. 15A and 15B show thatINSP005b-eletrotransferred animals show a decrease in transaminaseslevels as compared to empty vector control animals 8 hours after theConA challenge. In addition both TNF-alpha and IL-6 cytokine levels aresignificantly reduced in these animals (FIGS. 16A and 16B). The effectis similar to that obtained with the positive control vectorpDEST12.2hIL-6-SII.

4.6 Conclusion

These results show that delivery of INSP005b cDNA in an in vivo model offulminant hepatitis decreases TNF-alpha and m-IL-6 levels in serum andhad a significant effect on the reduction of ASAT and ALAT levelsmeasured in serum.

The decrease in ASAT and ALAT levels might be due to the decreasedTNF-alpha and IL-6 levels. TNF-alpha is an important cytokine involvedin the liver damage after ConA injection. In this mouse model of liverhepatitis TNF-alpha is mainly produced by hepatic macrophages, theso-called Kupfer cells. Anti TNF-alpha antibodies confer protectionagainst disease (Seino et al. 2001, Annals of surgery 234, 681).

1. An isolated polypeptide selected from the group consisting of: a) apolypeptide comprising SEQ ID NO: 34; b) a polypeptide consisting of SEQID NO: 34; c) a polypeptide having at least 97% sequence identity to SEQID NO: 34 and having metalloprotease activity; and d) a fusion proteincomprising a heterologous sequence fused to: SEQ ID NO: 34; or apolypeptide having at least 97% sequence identity to SEQ ID NO: 34 andhaving metalloprotease activity.
 2. The isolated polypeptide accordingto claim 1, wherein said polypeptide comprises SEQ ID NO:
 34. 3. Theisolated polypeptide according to claim 1, wherein said polypeptideconsists of SEQ ID NO:
 34. 4. The isolated polypeptide according toclaim 1, wherein said polypeptide has at least 97% sequence identity toSEQ ID NO: 34 and has metalloprotease activity.
 5. The isolatedpolypeptide according to claim 1, wherein said polypeptide is a fusionprotein comprising a heterologous sequence fused to: SEQ ID NO: 34; or apolypeptide having at least 97% sequence identity to SEQ ID NO: 34 andhaving metalloprotease activity.
 6. The isolated polypeptide accordingto claim 5, wherein said polypeptide is a fusion protein comprising aheterologous sequence fused to SEQ ID NO:
 34. 7. The isolatedpolypeptide according to claim 5, wherein said polypeptide is a fusionprotein comprising a heterologous sequence fused to a polypeptide havingat least 97% sequence identity to SEQ ID NO: 34 and havingmetalloprotease activity.
 8. The isolated polypeptide according to claim1, wherein said polypeptide has at least 98% sequence identity to SEQ IDNO: 34 and has metalloprotease activity.
 9. The isolated polypeptideaccording to claim 1, wherein said polypeptide is a fusion proteincomprising a heterologous sequence fused to a polypeptide having atleast 98% sequence identity to SEQ ID NO: 34 and having metalloproteaseactivity.
 10. The isolated polypeptide according to claim 1, whereinsaid polypeptide has at least 99% sequence identity to SEQ ID NO: 34 andhas metalloprotease activity.
 11. The isolated polypeptide according toclaim 1, wherein said polypeptide is a fusion protein comprising aheterologous sequence fused to a polypeptide having at least 99%sequence identity to SEQ ID NO: 34 and having metalloprotease activity.12. A pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and a polypeptide selected from the group consistingof: a) a polypeptide comprising SEQ ID NO: 34; b) a polypeptideconsisting of SEQ ID NO: 34; c) a polypeptide having at least 97%sequence identity to SEQ ID NO: 34 and having metalloprotease activity;and d) a fusion protein comprising a heterologous sequence fused to: SEQID NO: 34; or a polypeptide having at least 97% sequence identity to SEQID NO: 34 and having metalloprotease activity.
 13. The pharmaceuticalcomposition according to claim 12, wherein said polypeptide comprisesSEQ ID NO:
 34. 14. The pharmaceutical composition according to claim 12,wherein said polypeptide consists of SEQ ID NO:
 34. 15. Thepharmaceutical composition according to claim 12, wherein saidpolypeptide has at least 97% sequence identity to SEQ ID NO: 34 and hasmetalloprotease activity.
 16. The pharmaceutical composition accordingto claim 12, wherein said polypeptide is a fusion protein comprising aheterologous sequence fused to: SEQ ID NO: 34; or a polypeptide havingat least 97% sequence identity to SEQ ID NO: 34 and havingmetalloprotease activity.
 17. The pharmaceutical composition accordingto claim 16, wherein said polypeptide is a fusion protein comprising aheterologous sequence fused to SEQ ID NO:
 34. 18. The pharmaceuticalcomposition according to claim 16, wherein said polypeptide is a fusionprotein comprising a heterologous sequence fused to a polypeptide havingat least 97% sequence identity to SEQ ID NO: 34 and havingmetalloprotease activity.
 19. The pharmaceutical composition accordingto claim 12, wherein said polypeptide has at least 98% sequence identityto SEQ ID NO: 34 and has metalloprotease activity.
 20. Thepharmaceutical composition according to claim 12, wherein saidpolypeptide is a fusion protein comprising a heterologous sequence fusedto a polypeptide having at least 98 % sequence identity to SEQ ID NO: 34and having metalloprotease activity.
 21. The pharmaceutical compositionaccording to claim 12, wherein said polypeptide has at least 99%sequence identity to SEQ ID NO: 34 and has metalloprotease activity. 22.The pharmaceutical composition according to claim 12, wherein saidpolypeptide is a fusion protein comprising a heterologous sequence fusedto a polypeptide having at least 99% sequence identity to SEQ ID NO: 34and having metalloprotease activity.
 23. An immunogenic compositioncomprising an adjuvant and a polypeptide selected from the groupconsisting of: a) a polypeptide comprising SEQ ID NO: 34; b) apolypeptide consisting of SEQ ID NO: 34; c) a polypeptide having atleast 97% sequence identity to SEQ ID NO: 34 and having metalloproteaseactivity; and d) a fusion protein comprising a heterologous sequencefused to: SEQ ID NO: 34; or a polypeptide having at least 97% sequenceidentity to SEQ ID NO: 34 and having metalloprotease activity.
 24. Theimmunogenic composition according to claim 23, wherein said polypeptidecomprises SEQ ID NO:
 34. 25. The immunogenic composition according toclaim 23, wherein said polypeptide consists of SEQ ID NO:
 34. 26. Theimmunogenic composition according to claim 23, wherein said polypeptidehas at least 97% sequence identity to SEQ ID NO: 34 and hasmetalloprotease activity.
 27. The immunogenic composition according toclaim 23, wherein said polypeptide is a fusion protein comprising aheterologous sequence fused to: SEQ ID NO: 34; or a polypeptide havingat least 97% sequence identity to SEQ ID NO: 34 and havingmetalloprotease activity.
 28. The immunogenic composition according toclaim 27, wherein said polypeptide is a fusion protein comprising aheterologous sequence fused to SEQ ID NO:
 34. 29. The immunogeniccomposition according to claim 27, wherein said polypeptide is a fusionprotein comprising a heterologous sequence fused to a polypeptide havingat least 97% sequence identity to SEQ ID NO: 34 and havingmetalloprotease activity.
 30. The immunogenic composition according toclaim 23, wherein said polypeptide has at least 98% sequence identity toSEQ ID NO: 34 and has metalloprotease activity.
 31. The immunogeniccomposition according to claim 23, wherein said polypeptide is a fusionprotein comprising a heterologous sequence fused to a polypeptide havingat least 98% sequence identity to SEQ ID NO: 34 and havingmetalloprotease activity.
 32. The immunogenic composition according toclaim 23, wherein said polypeptide has at least 99% sequence identity toSEQ ID NO: 34 and has metalloprotease activity.
 33. The immunogeniccomposition according to claim 23, wherein said polypeptide is a fusionprotein comprising a heterologous sequence fused to a polypeptide havingat least 99% sequence identity to SEQ ID NO: 34 and havingmetalloprotease activity.
 34. A method of treating viral or acute liverdisease comprising administering, to an individual having viral or acuteliver disease, a pharmaceutical composition comprising a carrier and apolypeptide selected from the group consisting of: a) a polypeptidecomprising SEQ ID NO: 34; b) a polypeptide consisting of SEQ ID NO: 34;c) a polypeptide having at least 97% sequence identity to SEQ ID NO: 34and having metalloprotease activity; and d) a fusion protein comprisinga heterologous sequence fused to: SEQ ID NO: 34; or a polypeptide havingat least 97% sequence identity to SEQ ID NO: 34 and havingmetalloprotease activity; wherein said pharmaceutical composition isadministered in an amount effective to treat said viral or acute liverdisease.
 35. The method according to claim 34, wherein said polypeptidecomprises SEQ ID NO:
 34. 36. The method according to claim 34, whereinsaid polypeptide consists of SEQ ID NO:
 34. 37. The method according toclaim 34, wherein said polypeptide has at least 97% sequence identity toSEQ ID NO: 34 and has metalloprotease activity.
 38. The method accordingto claim 34, wherein said polypeptide is a fusion protein comprising aheterologous sequence fused to: SEQ ID NO: 34; or a polypeptide havingat least 97% sequence identity to SEQ ID NO: 34 and havingmetalloprotease activity.
 39. The method according to claim 38, whereinsaid polypeptide is a fusion protein comprising a heterologous sequencefused to SEQ ID NO:
 34. 40. The method according to claim 38, whereinsaid polypeptide is a fusion protein comprising a heterologous sequencefused to a polypeptide having at least 97% sequence identity to SEQ IDNO: 34 and having metalloprotease activity.
 41. The method according toclaim 34, wherein said acute liver disease is alcoholic liver failure.42. The method according to claim 34, wherein said polypeptide has atleast 98% sequence identity to SEQ ID NO: 34 and has metalloproteaseactivity.
 43. The method according to claim 34, wherein said polypeptideis a fusion protein comprising a heterologous sequence fused to apolypeptide having at least 98% sequence identity to SEQ ID NO: 34 andhaving metalloprotease activity.
 44. The method according to claim 34,wherein said polypeptide has at least 99% sequence identity to SEQ IDNO: 34 and has metalloprotease activity.
 45. The method according toclaim 34, wherein said polypeptide is a fusion protein comprising aheterologous sequence fused to a polypeptide having at least 99%sequence identity to SEQ ID NO: 34 and having metalloprotease activity.